Cell-penetrating compstatin analogs and uses thereof

ABSTRACT

In some aspects, the present invention cell-penetrating compstatin analog and compositions comprising cell-penetrating compstatin analog. In some aspects, the invention further provides methods of using cell-penetrating compstatin analogs treat a complement-mediated disorder. e.g., to inhibit complement-mediated damage to a cell, tissue, or organ, to inhibit production or release of biologically active C3 cleavage products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/791,631 filed Mar. 15, 2013, the entire contents of whichare hereby incorporated by reference.

BACKGROUND

Complement is a system consisting of more than 30 plasma and cell-boundproteins that plays a significant role in both innate and adaptiveimmunity. The proteins of the complement system act in a series ofenzymatic cascades through a variety of protein interactions andcleavage events. Complement activation occurs via three main pathways:the antibody-dependent classical pathway, the alternative pathway, andthe mannose-binding lectin (MBL) pathway. Inappropriate or excessivecomplement activation is an underlying cause or contributing factor to anumber of serious diseases and conditions, and considerable effort hasbeen devoted over the past several decades to exploring variouscomplement inhibitors as therapeutic agents. However, there remains aneed for innovative approaches to inhibiting complement activation for avariety of therapeutic purposes.

SUMMARY

In some aspects, the invention provides cell-penetrating compstatinanalogs. In some aspects, the invention provides methods of making,identifying, characterizing, and/or using cell-penetrating compstatinanalogs. In some aspects the invention provides compositions comprisingcell-penetrating compstatin analogs. In some aspects, the inventionprovides a physiologically acceptable composition comprising acell-penetrating compstatin analog. In some aspects, the inventionprovides a pharmaceutical grade composition comprising acell-penetrating compstatin analog.

In some aspects, the invention provides a composition comprising anisolated cell, tissue, or organ, and a cell-penetrating compstatinanalog. In some embodiments the invention provides an isolated cellcomprising a cell-penetrating compstatin analog inside the cell. In someembodiments the invention provides an isolated tissue or organ, whereinat least some of the cells of the tissue or organ comprise acell-penetrating compstatin analog inside them.

In some aspects, the invention provides methods of protecting a cell,tissue, organ, or subject from one or more effects of complement. Insome embodiments, the methods comprise contacting the cell, tissue, ororgan with a cell-penetrating compstatin analog. In some embodiments acell is contacted with a cell-penetrating compstatin analog for a timeand under conditions sufficient for internalization of thecell-penetrating compstatin analog by the cell. In some embodiments atissue or organ is contacted with a cell-penetrating compstatin analogfor a time and under conditions sufficient for internalization of thecell-penetrating compstatin analog by at least some cells of the tissueor organ. In some embodiments the cell, tissue, organ, or subject is theprotected from one or more effects of primate complement, e.g., humancomplement. In some embodiments the cell, tissue, organ, or subject isthe protected from one or more effects resulting from activation ofcomplement component C3, e.g., primate complement component C3, e.g.,human complement component C3.

A cell can be any type of cell in certain embodiments of any aspect ofthe invention. In some embodiments the cell is of a cell type thatnormally produces C3 in healthy subjects. In some embodiments the cellis of a cell type that exhibits increased C3 production and/or increasedC3 secretion in subjects suffering from or at risk of acomplement-mediated disease as compared with subjects not suffering fromor at risk of such disease. In some embodiments the cell is of a celltype that exhibits increased C3 production and/or increased C3secretion, e.g., in response to a stimulus. In some embodiments the cellis of a cell type that exhibits increased C3a production and/orincreased C3a secretion, e.g., in response to a stimulus. In someembodiments the cell is of a cell type that exhibits increased C3bproduction and/or increased C3b secretion, e.g., in response to astimulus. In some embodiments the stimulus is a stimulus that may causeor contribute to development or progression of a disease ormanifestation of a disease, e.g., a complement-mediated disease. In someembodiments, the cell is an immune system cell. In some embodiments, thecell is a lymphoid cell. In some embodiments, the cell is a myeloidcell. In some embodiments, the cell is an epithelial cell. In someembodiments, the cell is a respiratory epithelial cell. In someembodiments, the cell is a retinal pigment epithelial cell. In someembodiments the cell is an endothelial cell. In some embodiments thecell is a nervous system cell, e.g., a neuron or glial cell.

In some embodiments, a cell, tissue, or organ is contacted ex vivo(outside the body of a subject). In some embodiments a cell, tissue, ororgan is contacted in vivo (in a subject, e.g., a human). In someembodiments a cell, tissue, or organ is to be transplanted into asubject or has been transplanted into a subject. In some embodiments acell-penetrating compstatin analog is administered to a subject.

In some aspects the invention provides methods of treating a subject inneed of treatment for a complement-mediated disorder. In someembodiments the methods comprise administering a cell-penetratingcompstatin analog to the subject.

All articles, books, patent applications, patents, other publications,websites, and databases mentioned in this application are incorporatedherein by reference. In the event of a conflict between thespecification and any of the incorporated references the specification(including any amendments thereto) shall control. Unless otherwiseindicated, art-accepted meanings of terms and abbreviations are usedherein. The practice of certain aspects described herein may employconventional techniques of molecular biology, cell culture, recombinantnucleic acid (e.g., DNA) technology, immunology, and/or nucleic acid andpolypeptide synthesis, detection, manipulation, and quantification,etc., that are within the ordinary skill of the art. See, e.g., Ausubel,F., et al, (eds.), Current Protocols in Molecular Biology, CurrentProtocols in Immunology, Current Protocols in Protein Science, andCurrent Protocols in Cell Biology, all John Wiley & Sons, N.Y., e.g.,edition current as of January 2010 or later; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001 or 4^(th) ed,2012, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 1988.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS I. Definitions

The terms “approximately” or “about” in reference to a number generallyinclude numbers that fall within ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments ±0.5% of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would impermissibly exceed 100% of a possible value).

A “complement component” or “complement protein” is a protein that isinvolved in activation of the complement system or participates in oneor more complement-mediated activities. Components of the classicalcomplement pathway include, e.g., C1q, C1r, C1s, C2, C3, C4, C5, C6, C7,C8, C9, and the C5b-9 complex, also referred to as the membrane attackcomplex (MAC) and active fragments or enzymatic cleavage products of anyof the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components ofthe alternative pathway include, e.g., factors B, D, and properdin.Components of the lectin pathway include, e.g., MBL2, MASP-1, andMASP-2. Complement components also include cell-bound receptors forsoluble complement components, wherein such receptor mediates one ormore biological activities of such soluble complement componentfollowing binding of the soluble complement component. Such receptorsinclude, e.g., C5a receptor (C5aR), C3a receptor (C3aR), ComplementReceptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3(CR3, also known as CD45), etc. It will be appreciated that the term“complement component” is not intended to include those molecules andmolecular structures that serve as “triggers” for complement activation,e.g., antigen-antibody complexes, foreign structures found on microbialor artificial surfaces, etc.

A “complement-mediated disorder” is any disorder in which complementactivation is known or suspected of being a contributing and/or at leastpartially causative factor in at least some subjects suffering from thedisorder, e.g., disorders in which complement activation results intissue damage. Non-limiting examples of complement-mediated disordersinclude, but are not limited to, (i) various disorders characterized byhemolysis or hemolytic anemia such as atypical hemolytic uremicsyndrome, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria,transfusion reactions; (ii) transplant rejection (e.g., hyperacute oracute transplant rejection) or transplant dysfunction; (iii) disordersinvolving ischemia/reperfusion injury such as trauma, surgery (e.g.,aneurysm repair), myocardial infarction, ischemic stroke; (iv) disordersof the respiratory system such as asthma and chronic obstructivepulmonary disease (COPD); (v) arthritis, e.g., rheumatoid arthritis;(vi) ocular disorders such as age-related macular degeneration (AMD),diabetic retinopathy, glaucoma, and uveitis. “Disorder” is usedinterchangeably herein with “disease”, “condition”, and similar words torefer to any impairment of health or state of abnormal functioning of anorganism, e.g., any state in which medical and/or surgical management isindicated or for which a subject appropriately seeks medical and/orsurgical attention. It should also be understood that the listing of aparticular disorder within a particular category is for convenience andis not intended to limit the invention. It will be understood thatcertain disorders could appropriately be listed in multiple categories.

A “complement regulatory protein” is a protein involved in regulatingcomplement activity. A complement regulatory protein may down-regulatecomplement activity by, e.g., inhibiting complement activation or byinactivating or accelerating decay of one or more activated complementproteins. Examples of complement regulatory proteins include CIinhibitor, C4 binding protein, clusterin, vitronectin, CFH, factor I,and the cell-bound proteins CD46, CD55, CD59, CR1, CR2, and CR3.

“Isolated”, as used herein, means 1) separated from at least some of thecomponents with which it is usually associated in nature; 2) prepared orpurified by a process that involves the hand of man; and/or 3) notoccurring in nature, e.g., present in an artificial environment. Ingeneral, unless otherwise indicated or clearly evident, any entity,product, agent, composition, etc., may be deemed “isolated”, if desired.

“Linked”, as used herein with respect to two or more moieties, meansthat the moieties are physically associated or connected with oneanother to form a molecular structure that is sufficiently stable sothat the moieties remain associated under the conditions in which thelinkage is formed and, preferably, under the conditions in which the newmolecular structure is used, e.g., physiological conditions. In certainpreferred embodiments of the invention the linkage is a covalentlinkage. In other embodiments the linkage is noncovalent. Moieties maybe linked either directly or indirectly. When two moieties are directlylinked, they are either covalently bonded to one another or are insufficiently close proximity such that intermolecular forces between thetwo moieties maintain their association. When two moieties areindirectly linked, they are each linked either covalently ornoncovalently to a third moiety, which maintains the association betweenthe two moieties. In general, when two moieties are referred to as beinglinked by a “linking moiety” or “linking portion”, the linkage betweenthe two linked moieties is indirect, and typically each of the linkedmoieties is covalently bonded to the linking moiety. Two moieties may belinked using a “linker”. A linker can be any suitable moiety that reactswith the entities to be linked within a reasonable period of time, underconditions consistent with stability of the entities (portions of whichmay be protected as appropriate, depending upon the conditions), and insufficient amount, to produce a reasonable yield. Typically the linkerwill contain at least two functional groups, one of which reacts with afirst entity and the other of which reacts with a second entity. It willbe appreciated that after the linker has reacted with the entities to belinked, the term “linker” may refer to the part of the resultingstructure that originated from the linker, or at least the portion thatdoes not include the reacted functional groups. A linking moiety maycomprise a portion that does not participate in a bond with the entitiesbeing linked, and whose main purpose may be to spatially separate theentities from each other. Such portion may be referred to as a “spacer”.

As used herein, “physiological conditions” refers to a set of conditionssuch as temperature, salt concentration, pH that at least in part mimicthose conditions as typically found in a living subject, e.g., amammalian subject. In some aspects, physiological conditions refer toconditions in an aqueous medium, e.g., a medium comprising at least 90%,95%, 96%, 97%, 97%, 99%, or about 100% water on a volume/volume basis.In some embodiments other liquids, if present, do not substantiallyaffect protein secondary or tertiary structure. In some embodimentsphysiological conditions at least in part mimic those found in a bodyfluid such as blood or extracellular fluid, e.g., interstitial fluid,e.g., of a mammalian subject. A variety of physiological conditionsuseful for, e.g., in vitro assays, are known in the art. Generally, amedium under physiological conditions contains a physiologicalconcentration of salt, e.g., sodium chloride. In some embodiments aphysiological concentration of salt refers to a concentration rangingfrom about 250 mOsm/L to about 350 mOsm/L, e.g., about 275 mOsm/L toabout 325 mOsm/L, e.g., about 300 mOsm/L. In some embodimentsphysiological conditions are approximately isotonic to a body fluid,e.g., blood or extracellular fluid, e.g., interstitial fluid. In someembodiments physiological conditions include a pH ranging from about 6.5to about 7.8, e.g., about 7.0 to about 7.5. In some embodiments aphysiological medium comprises a buffer substance that helps maintainthe pH of the medium within a physiological range. In some embodimentsphysiological conditions comprise conditions such that a typicalmammalian protein, e.g., a protein typically found in a body fluid, suchas blood or extracellular fluid, substantially retains the secondaryand, if applicable, tertiary structure that such protein has in the bodyfluid in which it is normally found. In some embodiments components of aphysiological medium are typically substantially non-toxic to mammaliancells at the concentration at which they are present in thephysiological medium. A variety of physiological media (sometimes termed“buffers”) are listed in various standard references, such as thosecited above (e.g., Sambrook, et al, Protocols series). In someembodiments a physiological temperature ranges from about 25 degrees C.to about 38 degrees C., e.g., from about 30 degrees C. to about 37degrees C., e.g., 35 degrees C. to 37 degrees C.

“Polypeptide”, as used herein, refers to a polymer of amino acids,optionally including one or more amino acid analogs. A protein is amolecule composed of one or more polypeptides. A peptide is a relativelyshort polypeptide, typically between about 2 and 60 amino acids inlength, e.g., between 8 and 40 amino acids in length. The terms“protein”, “polypeptide”, and “peptide” may be used interchangeably.Polypeptides used herein may contain amino acids such as those that arenaturally found in proteins, amino acids that are not naturally found inproteins, and/or amino acid analogs that are not amino acids. As usedherein, an “analog” of an amino acid may be a different amino acid thatstructurally resembles the amino acid or a compound other than an aminoacid that structurally resembles the amino acid. A large number ofart-recognized analogs of the 20 amino acids commonly found in proteins(the “standard” amino acids) are known. One or more of the amino acidsin a polypeptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Certainnon-limiting suitable analogs and modifications are described inWO2004026328 and/or below. The polypeptide may be acetylated, e.g., atthe N-terminus and/or amidated, e.g., at the C-terminus.

The term “purified”, as used herein, refers to substances that have beenseparated from at least some or most of the components with which theyare associated in nature or when originally generated or with which theywere associated prior to purification. In general, such purificationinvolves action of the hand of man. Purified agents may be partiallypurified, substantially purified, or pure. Such agents may be, forexample, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or more than 99% pure. In some embodiments, a nucleic acid,polypeptide, or small molecule is purified such that it constitutes atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of the totalnucleic acid, polypeptide, or small molecule material, respectively,present in a preparation. In some embodiments, an organic substance,e.g., a nucleic acid, polypeptide, or small molecule, is purified suchthat it constitutes at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or more, of the total organic material present in a preparation.Purity may be based on, e.g., dry weight, size of peaks on achromatography tracing (GC, HPLC, etc.), molecular abundance,electrophoretic methods, intensity of bands on a gel, spectroscopic data(e.g., NMR), elemental analysis, high throughput sequencing, massspectrometry, or any art-accepted quantification method. In someembodiments, water, buffer substances, ions, and/or small molecules(e.g., synthetic precursors such as nucleotides or amino acids), canoptionally be present in a purified preparation. A purified agent may beprepared by separating it from other substances (e.g., other cellularmaterials), or by producing it in such a manner to achieve a desireddegree of purity. In some embodiments “partially purified” with respectto a molecule produced by a cell means that a molecule produced by acell is no longer present within the cell, e.g., the cell has been lysedand, optionally, at least some of the cellular material (e.g., cellwall, cell membrane(s), cell organelle(s)) has been removed and/or themolecule has been separated or segregated from at least some moleculesof the same type (protein, RNA, DNA, etc.) that were present in thelysate.

“Recombinant host cells”, “host cells”, and other such terms, denoteprokaryotic or eukaryotic cells or cell lines that contain an exogenousnucleic acid (typically DNA) such as an expression vector comprising anucleic acid that encodes a polypeptide of interest. It will beunderstood that such terms include the descendants of the originalcell(s) into which the vector or other nucleic acid has been introduced.Appropriate host cells include any of those routinely used in the artfor expressing polynucleotides (e.g., for purposes of producingpolypeptide(s) encoded by such polynucleotides) including, for example,prokaryotes, such as E. coli or other bacteria such as species ofEscherichia; Lactobacillus, Bacillus (e.g., B subtilis), SalmonellaPseudomonas, Streptomyces, Staphylococcus, etc; and eukaryotes,including for example, fungi, such as yeast (e.g., Pichia (e.g., Pichiapastoris), Kluyveromyces, such as K. lactis, Hansenula, e.g. H.polymorpha). Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. orTrichoderma spp., e.g., strains of A. oryzae, A. nidulans or A. niger;insect cells (e.g., Sf9), plant cells, and animal cells, e.g., mammaliancells such as CHO, R1.1, B-W, L-M, African Green Monkey Kidney cells(e.g. COS-1, COS-7, BSC-1, BSC-40 and BMT-10), and cultured human cells.Also encompassed are genetically modified cells in genetically modified(e.g., transgenic) plants or animals, wherein a recombinant polypeptideis produced by at least some such cells. A polypeptide may be secretedin milk, harvested from plant material, etc. The exogenous nucleic acidmay be stably maintained as an episome such as a plasmid or may at leastin part be integrated into the host cell's genome, optionally afterbeing copied or reverse transcribed. Terms such as “host cells”, etc.,are also used to refer to cells or cell lines that can be used asrecipients for an exogenous nucleic acid, prior to introduction of thenucleic acid. A “recombinant polynucleotide” generally is apolynucleotide that contains nucleic acid sequences that are not foundjoined directly to one another in nature. For example, the nucleic acidsequences may occur in different genes or different species or one ormore of the sequence(s) may be a variant of a naturally occurringsequence or may at least in part be an artificial sequence that is nothomologous to a naturally occurring sequence. A “recombinantpolypeptide” generally is a polypeptide that is at least in partproduced by transcription and translation of an exogenous nucleic acidby a recombinant host cell or by a cell-free in vitro expression systemand/or that contains amino acid sequences that are not found joineddirectly to one another in nature. In the latter case, the recombinantpolypeptide may be referred to as a “chimeric polypeptide”. The aminoacid sequences in a chimeric polypeptide may, for example, occur indifferent genes or in different species or one or more of thesequence(s) may be a variant of a naturally occurring sequence or may atleast in part be an artificial sequence that is not identical or in someembodiments is not homologous to a naturally occurring sequence over asubstantial portion of the length. It will be understood that a chimericpolypeptide may comprise two or more polypeptides. For example, firstand second polypeptides A and B of a chimeric polypeptide may bedirectly linked (A-B or B-A) or may be separated by a third polypeptideportion C (A-C-B or B-C-A). In some embodiments, portion C represents apolypeptide linker which may, for example, comprise multiple glycineand/or serine residues or any of a variety of other amino acids. In someembodiments, two or more polypeptides may be linked by non-polypeptidelinker(s). “Recombinant” as used herein encompasses in certainembodiments polypeptides produced by joining (e.g., chemicallyconjugating, enzymatically conjugating), shorter recombinantpolypeptides that may be produced in recombinant host cells. In someembodiments a recombinant polypeptide may comprise a signal sequencethat directs secretion of the polypeptide or a sequence that directs theexpressed polypeptide to a specific compartment or organelle. Suitablesequences are known in the art. Appropriate sequences for a host celltype of interest (e.g., bacterial, fungal, mammalian, plant, etc.) maybe selected. A signal sequence may be located at or near (e.g., withinup to 10-50 amino acids of) the N-terminus or C-terminus in someembodiments. In some embodiments a polypeptide comprises a tag. A tagmay be useful to facilitate detection and/or purification of a proteinthat contains it. Examples of tags include polyhistidine-tag (e.g.,6×-His tag), glutathione-S-transferase, maltose binding protein, NUStag, SNUT tag, Strep tag, epitope tags such as V5, HA, Myc, or FLAG. Insome embodiments a protease cleavage site is located in the regionbetween the tag and the polypeptide, allowing the polypeptide to beseparated from the tag by exposure to the protease. In some embodimentsa polynucleotide that encodes a recombinant polypeptide is at least inpart codon optimized for expression in a host cell of interest (e.g.,bacterial, fungal, mammalian, plant, etc.). A tag may be located at ornear (e.g., within up to 10-50 amino acids of) the N- or C-terminus of apolypeptide in various embodiments. A recombinant polypeptide may beisolated, purified, etc., using any of a variety of methods. See, e.g.,Sambrook, Protocols series, or other standard references. Methods of usemay include, e.g., dialysis (e.g., using membranes having defined poresize), chromatography, precipitation, gel purification, oraffinity-based methods that may, in some embodiments, utilize a tag or aspecific binding reagent such as an antibody.

“Reactive functional groups” as used herein refers to groups including,but not limited to, olefins, acetylenes, alcohols, phenols, ethers,oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides,cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acidsisonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds, N-hydroxysuccinimide esters,maleimides, sulfhydryls, and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989, andHermanson, G., Bioconjugate Techniques, 2^(nd) ed., Academic Press, SanDiego, 2008).

“Specific binding” generally refers to a physical association between atarget polypeptide (or, more generally, a target molecule) and a bindingmolecule such as an antibody or ligand. The association is typicallydependent upon the presence of a particular structural feature of thetarget such as an antigenic determinant, epitope, binding pocket orcleft, recognized by the binding molecule. For example, if an antibodyis specific for epitope A, the presence of a polypeptide containingepitope A or the presence of free unlabeled A in a reaction containingboth free labeled A and the binding molecule that binds thereto, willreduce the amount of labeled A that binds to the binding molecule. It isto be understood that specificity need not be absolute but generallyrefers to the context in which the binding occurs. For example, it iswell known in the art that numerous antibodies cross-react with otherepitopes in addition to those present in the target molecule. Suchcross-reactivity may be acceptable depending upon the application forwhich the antibody is to be used. One of ordinary skill in the art willbe able to select antibodies or ligands having a sufficient degree ofspecificity to perform appropriately in any given application (e.g., fordetection of a target molecule, for therapeutic purposes, etc). It isalso to be understood that specificity may be evaluated in the contextof additional factors such as the affinity of the binding molecule forthe target versus the affinity of the binding molecule for othertargets, e.g., competitors. If a binding molecule exhibits a highaffinity for a target molecule that it is desired to detect and lowaffinity for nontarget molecules, the antibody will likely be anacceptable reagent. Once the specificity of a binding molecule isestablished in one or more contexts, it may be employed in other,preferably similar, contexts without necessarily re-evaluating itsspecificity. In some embodiments, the affinity (as measured by theequilibrium dissociation constant, Kd) of two molecules that exhibitspecific binding is 10⁻³ M or less, e.g., 10⁻⁴ M or less, e.g., 10⁻⁵ Mor less, e.g., 10⁻⁶ M or less, 10⁻⁷M or less, 10⁻⁸M or less, or 10⁻⁹ Mor less under the conditions tested, e.g., under physiologicalconditions.

A “subject” treated according to the instant invention is typically ahuman, a non-human primate, or a lower animal (e.g., a mouse or rat),which expresses or contains at least some primate (e.g., human)complement component C3 and, optionally, one or more additional primatecomplement component(s). In some embodiments the subject is male. Insome embodiments the subject is female. In some embodiments the subjectis an adult, e.g., a human at least 18 years of age, e.g., between 18and 100 years of age. In some embodiments, a human subject is at least12 years of age. In some embodiments a subject is an adult, e.g., ahuman at least 18 years of age, e.g., between 18 and 100 years of age.In some embodiments a subject is at least 40, 45, 50, 55, 60, 65, 70,75, or 80 years of age. In some embodiments the subject is a child,e.g., a human between 0 and 4 years of age, or between 5 and 11 years ofage.

“Treating”, as used herein in regard to treating a subject, refers toproviding treatment, i.e., providing any type of medical or surgicalmanagement of a subject. The treatment can be provided in order toreverse, alleviate, inhibit the progression of, prevent or reduce thelikelihood of a disease, or in order to reverse, alleviate, inhibit orprevent the progression of, prevent or reduce the likelihood of one ormore symptoms or manifestations of a disease. “Prevent” refers tocausing a disease or symptom or manifestation of a disease not to occurfor at least a period of time in at least some individuals. Treating caninclude administering a compound or composition to the subject followingthe development of one or more symptoms or manifestations indicative ofa disease, e.g., in order to reverse, alleviate, reduce the severity of,and/or inhibit or prevent the progression of the disease and/or toreverse, alleviate, reduce the severity of, and/or inhibit or one ormore symptoms or manifestations of the disease. A compound orcomposition can be administered to a subject who has developed adisease, or is at increased risk of developing the disease relative to amember of the general population. A compound or composition can beadministered to a subject who has developed a disease and is atincreased risk of developing one or more particular symptoms ormanifestations of the disease or an exacerbation of the disease relativeto other individuals diagnosed with the disease, or relative to thesubject's typical or average risk for such symptom or manifestation orexacerbation. For example, the subject may have been exposed to a“trigger” that places the subject at increased risk (e.g., temporarilyincreased risk) of experiencing an exacerbation. A compound orcomposition can be administered prophylactically, i.e., beforedevelopment of any symptom or manifestation of the disease. Typically inthis case the subject will be at risk of developing the disease, e.g.,relative to a member of the general population, optionally matched interms of age, sex, and/or other demographic variable(s).

A “vector” may be any of a variety of nucleic acid molecules, viruses,or portions thereof that are capable of mediating entry of, e.g.,transferring, transporting, etc., a nucleic acid of interest betweendifferent genetic environments or into a cell. The nucleic acid ofinterest may be linked to, e.g., inserted into, the vector using, e.g.,restriction and ligation. Vectors include, for example, DNA or RNAplasmids, cosmids, naturally occurring or modified viral genomes orportions thereof, nucleic acids that can be packaged into viral capsids,mini-chromosomes, artificial chromosomes, etc. Plasmid vectors typicallyinclude an origin of replication (e.g., for replication in prokaryoticcells). A plasmid may include part or all of a viral genome (e.g., aviral promoter, enhancer, processing or packaging signals, and/orsequences sufficient to give rise to a nucleic acid that can beintegrated into the host cell genome and/or to give rise to infectiousvirus). Viruses or portions thereof that can be used to introducenucleic acids into cells may be referred to as viral vectors. Viralvectors include, e.g., adenoviruses, adeno-associated viruses,retroviruses (e.g., lentiviruses, vaccinia virus and other poxviruses,herpesviruses (e.g., herpes simplex virus), and others. Baculovirus areof use, e.g., in insect cells. A wide range of plant viral vectors areknown and include, e.g., those based on or comprising Cauliflower MosaicVirus, Tobacco Mosaic Virus, or one or more genetic elements thereof(e.g., Cauliflower Mosaic Virus 35S promoter). Viral vectors may or maynot contain sufficient viral genetic information for production ofinfectious virus when introduced into host cells, i.e., viral vectorsmay be replication-competent or replication-defective. In someembodiments, e.g., where sufficient information for production ofinfectious virus is lacking, it may be supplied by a host cell or byanother vector introduced into the cell, e.g., if production of virus isdesired. In some embodiments such information is not supplied, e.g., ifproduction of virus is not desired. A nucleic acid to be transferred maybe incorporated into a naturally occurring or modified viral genome or aportion thereof or may be present within a viral capsid as a separatenucleic acid molecule. A vector may contain one or more nucleic acidsencoding a marker suitable for identifying and/or selecting cells thathave taken up the vector. Markers include, for example, various proteinsthat increase or decrease either resistance or sensitivity toantibiotics or other agents (e.g., a protein that confers resistance toan antibiotic such as puromycin, hygromycin or blasticidin), enzymeswhose activities are detectable by assays known in the art (e.g.,β-galactosidase or alkaline phosphatase), and proteins or RNAs thatdetectably affect the phenotype of cells that express them (e.g.,fluorescent proteins). Vectors often include one or more appropriatelypositioned sites for restriction enzymes, which may be used tofacilitate insertion into the vector of a nucleic acid, e.g., a nucleicacid to be expressed. An expression vector is a vector into which adesired nucleic acid has been inserted or may be inserted such that itis operably linked to regulatory elements (also termed “regulatorysequences”, “expression control elements”, or “expression controlsequences”) and may be expressed as an RNA transcript (e.g., an mRNAthat can be translated into protein or a noncoding RNA). Expressionvectors include regulatory sequence(s), e.g., expression controlsequences, sufficient to direct transcription of an operably linkednucleic acid under at least some conditions; other elements required orhelpful for expression may be supplied by, e.g., the host cell or by anin vitro expression system. Such regulatory sequences typically includea promoter and may include enhancer sequences or upstream activatorsequences. In some embodiments a vector may include sequences thatencode a 5′ untranslated region and/or a 3′ untranslated region, whichmay comprise a cleavage and/or polyadenylation signal. In general,regulatory elements may be contained in a vector prior to insertion of anucleic acid whose expression is desired or may be contained in aninserted nucleic acid or may be inserted into a vector followinginsertion of a nucleic acid whose expression is desired. As used herein,a nucleic acid and regulatory element(s) are said to be “operablylinked” when they are covalently linked so as to place the expression ortranscription of the nucleic acid under the influence or control of theregulatory element(s). For example, a promoter region would be operablylinked to a nucleic acid if the promoter region were capable ofeffecting transcription of that nucleic acid. One of ordinary skill inthe art will be aware that the precise nature of the regulatorysequences useful for gene expression may vary between species or celltypes, but may in general include, as appropriate, sequences involvedwith the initiation of transcription, RNA processing, or initiation oftranslation. The choice and design of an appropriate vector andregulatory element(s) is within the ability and discretion of one ofordinary skill in the art. For example, one of skill in the art willselect an appropriate promoter (or other expression control sequences)for expression in a desired species (e.g., a prokaryotic (bacterial) oreukaryotic (e.g., fungal, plant, mammalian species) or cell type. Avector may contain a promoter capable of directing expression inmammalian cells, such as a suitable viral promoter, e.g., from acytomegalovirus (CMV), retrovirus, simian virus (e.g., SV40), papillomavirus, herpes virus or other virus that infects mammalian cells, or amammalian promoter from, e.g., a gene such as EF1 alpha, ubiquitin(e.g., ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK),etc., or a composite promoter such as a CAG promoter (combination of theCMV early enhancer element and chicken beta-actin promoter). In someembodiments a human promoter may be used. In some embodiments, apromoter that ordinarily directs transcription by a eukaryotic RNApolymerase I (a “pol I promoter”), e.g., (a U6, HI, 7SK or tRNA promoteror a functional variant thereof) may be used. In some embodiments, apromoter that ordinarily directs transcription by a eukaryotic RNApolymerase II (a “pol II promoter”) or a functional variant thereof isused. In some embodiments, a promoter that ordinarily directstranscription by a eukaryotic RNA polymerase III (a “pol III promoter”),e.g., a promoter for transcription of ribosomal RNA (other than 5S rRNA)or a functional variant thereof is used. One of ordinary skill in theart will select an appropriate promoter for directing transcription of asequence of interest. Examples of expression vectors that may be used inmammalian cells include, e.g., the pcDNA vector series, pSV2 vectorseries, pCMV vector series, pRSV vector series, pEFI vector series,Gateway® vectors, etc. In some embodiments, regulatable (e.g., inducibleor repressible) expression control element(s), e.g., a regulatablepromoter, is/are used so that expression can be regulated, e.g., turnedon or increased or turned off or decreased. In some embodiments a vectormay comprise a polynucleotide sequence that encodes a polypeptide,wherein the polynucleotide sequence is positioned in frame with anucleic acid inserted into the vector so that an N- or C-terminal fusionis created. In some embodiments a polypeptide encoded by thepolynucleotide sequence may comprise a signal sequence (which directssecretion of a protein) or a sequence that directs the expressed proteinto a specific organelle or location in the cell such as the nucleus ormitochondria. In some embodiments a polypeptide comprises a tag. A tagmay be useful to facilitate detection and/or purification of a proteinthat contains it. Examples of tags include polyhistidine-tag (e.g.,6×-His tag), glutathione-S-transferase, maltose binding protein, NUStag, SNUT tag, Strep tag, epitope tags such as V5, HA, Myc, or FLAG. Insome embodiments a protease cleavage site is located in the regionbetween the protein encoded by the inserted nucleic acid and thepolypeptide, allowing the polypeptide to be removed by exposure to theprotease. Vectors may be introduced into host cells using methods knownin the art. One of ordinary skill will select an appropriate methodbased, e.g., ors the vector, cell type, etc. Examples of suitablemethods include, e.g., calcium phosphate-mediated transfection,transfection with any of a variety of commercially available reagents,e.g., lipid-based or non-lipid based, such as FuGENE, Lipofectamine,TurboFect; electroporation; microparticle bombardment, etc. Such methodsare explained in detail in standard references such as Sambrook,Protocols series, and others.

As used herein the term “aliphatic” denotes a hydrocarbon moiety thatmay be straight-chain (i.e., unbranched), branched, or cyclic (includingfused, bridging, and spiro-fused polycyclic) and may be completelysaturated or may contain one or more units of unsaturation, but which isnot aromatic. Unless otherwise specified, aliphatic groups contain 1-30carbon atoms. In some embodiments, aliphatic groups contain 1-10 carbonatoms. In other embodiments, aliphatic groups contain 1-8 carbon atoms.In still other embodiments, aliphatic groups contain 1-6 carbon atoms,and in yet other embodiments aliphatic groups contain 1{circumflex over( )}1 carbon atoms. Suitable aliphatic groups include, but are notlimited to, linear or branched, alkyl, alkenyl, and alkynyl groups, andhybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

As used herein, “alkyl” refers to a saturated straight, branched, orcyclic hydrocarbon having from about 1 to about 22 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 1 to about 12, or about 1 toabout 7 carbon atoms being preferred in certain embodiments of theinvention. Alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl,adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

As used herein, “halo” refers to F, C1, Br or I.

As used herein, “alkanoyl” refers to an optionally substituted straightor branched aliphatic acyclic residue having about 1 to 10 carbon atoms(and all combinations and subcombinations of ranges and specific numberof carbon atoms) therein, e.g., from about 1 to 7 carbon atoms which, aswill be appreciated, is attached to a terminal C=0 group with a singlebond (and may also be referred to as an “acyl group”). Alkanoyl groupsinclude, but are not limited to, formyl, acetyl, propionyl, butyryl,isobutyryl, pentanoyl, isopentanoyl, 2-methyl-butyryl,2,2-dimethoxypropionyl, hexanoyl, heptanoyl, octanoyl, and the like, andfor purposes of the present invention a formyl group is considered analkanoyl group. “Lower alkanoyl” refers to an optionally substitutedstraight or branched aliphatic acyclic residue having about 1 to about 5carbon atoms (and all combinations and subcombinations of ranges andspecific number of carbon atoms). Such groups include, but are notlimited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl,isopentanoyl, etc.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl radicals bearing an arylsubstituent and having from about 6 to about 22 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred in certain embodiments. Aralkyl groups can be optionallysubstituted. Non-limiting examples include, for example, benzyl,naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl, anddiphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O— group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy.

As used herein, “carboxy” refers to a —C(=0)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(=0)0-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(=0)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

The term “cyclic ring system” refers to an aromatic or non-aromatic,partially unsaturated or fully saturated, 3- to 10-membered ring system,which includes single rings of 3 to 8 atoms in size and bi- andtri-cyclic ring systems which may include aromatic 5- or 6-membered arylor aromatic heterocyclic groups fused to a non-aromatic ring. Theseheterocyclic rings include those having from 1 to 3 heteroatomsindependently selected from the group consisting of oxygen, sulfur, andnitrogen. In certain embodiments, the term heterocyclic refers to anon-aromatic 5-, 6-, or 7-membered ring or a polycyclic group wherein atleast one ring atom is a heteroatom selected from the group consistingof O, S. and N, including, but not limited to, a bi- or tri-cyclicgroup, comprising fused six-membered rings having between one and threeheteroatoms independently selected from the group consisting of theoxygen, sulfur, and nitrogen. In some embodiments, “cyclic ring system”refers to a cycloalkyl group which, as used herein, refers to groupshaving 3 to 10, e.g., 4 to 7 carbon atoms. Cycloalkyls include, but arenot limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and the like, which, is optionally substituted. In someembodiments, “cyclic ring system” refers to a cycloalkenyl orcycloalkynyl moiety, which is optionally substituted.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl(—OH), alkoxyl, cyano (—CN), carboxyl (—COOH), —C(=0)0-alkyl,aminocarbonyl (—C(=0)NH₂), —N-substituted aminocarbonyl (—C(=0)NHR″),CF₃, CF₂CF₃, and the like. In relation to the aforementionedsubstituents, each moiety R″ can be, independently, any of H, alkyl,cycloalkyl, aryl, or aralkyl, for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term “D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

As used herein, an “aromatic amino acid” is an amino acid that comprisesat least one aromatic ring, e.g., it comprises an aryl group.

As used herein, an “aromatic amino acid analog” is an amino acid analogthat comprises at least one aromatic ring, e.g., it comprises an arylgroup.

II. Overview

In some aspects, the invention provides cell-penetrating compstatinanalogs. Cell-penetrating compstatin analogs (CPCA) are compounds thatcomprise a compstatin analog moiety and a cell penetrating moiety (CPM),wherein the cell penetrating moiety is capable of enhancing entry of thecompound into cells as compared to entry of a compstatin analog of thesame sequence but lacking the cell penetrating moiety. A CPCA is thusable to enter the cell in increased amounts relative to the compstatinanalog of the same sequence but lacking the cell penetrating moiety.Without wishing to be bound by any theory, a cell-penetrating compstatinanalog may (i) bind to C3 within the cell and inhibit its activationwithin the cell; bind to C3 or C3b within the cell and inhibit itssecretion; and/or (iii) bind to C3 or C3b within the cell and thusinhibit activity or activation of C3 or C3b that is subsequentlysecreted by the cell. C3 or C3b bound to a CPCA may thus be inhibitedfrom participation in the complement activation cascade.

In some aspects, the invention provides methods of makingcell-penetrating compstatin analogs. In some aspects, the inventionprovides methods of characterizing cell-penetrating compstatin analogs.In some aspects, the invention provides methods of usingcell-penetrating compstatin analogs. In some aspects, the inventionprovides compositions comprising cell-penetrating compstatin analogs.

III. Complement System

In order to facilitate understanding of the invention, and withoutintending to limit the invention in any way, this section provides anoverview of complement and its pathways of activation. Further detailsare found, e.g., in Kuby Immunology, 6^(th) ed., 2006; Paul, W. E.,Fundamental Immunology, Lippincott Williams & Wilkins; 6^(th) ed., 2008;and Walport M J., Complement. First of two parts. N Engl J Med.,344(14): 1058-66, 2001.

Complement is an arm of the innate immune system that plays an importantrole in defending the body against infectious agents. The complementsystem comprises more than 30 serum and cellular proteins that areinvolved in three major pathways, known as the classical, alternative,and lectin pathways. The classical pathway is usually triggered bybinding of a complex of antigen and IgM or IgG antibody to CI (thoughcertain other activators can also initiate the pathway). Activated CIcleaves C4 and C2 to produce C4a and C4b, in addition to C2a and C2b.C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3aand C3b. Binding of C3b to C3 convertase produces C5 convertase, whichcleaves C5 into C5a and C5b. C3a, C4a, and C5a are anaphylotoxins andmediate multiple reactions in the acute inflammatory response. C3a andC5a are also chemotactic factors that attract immune system cells suchas neutrophils.

The alternative pathway is initiated by and amplified at, e.g.,microbial surfaces and various complex polysaccharides. In this pathway,hydrolysis of C3 to C3(H20), which occurs spontaneously at a low level,leads to binding of factor B, which is cleaved by factor D, generating afluid phase C3 convertase that activates complement by cleaving C3 intoC3a and C3b. C3b binds to targets such as cell surfaces and forms acomplex with factor B, which is later cleaved by factor D, resulting ina C3 convertase. Surface-bound C3 convertases cleave and activateadditional C3 molecules, resulting in rapid C3b deposition in closeproximity to the site of activation and leading to formation ofadditional C3 convertase, which in turn generates additional C3b. Thisprocess results in a cycle of C3 cleavage and C3 convertase formationthat significantly amplifies the response. Cleavage of C3 and binding ofanother molecule of C3b to the C3 convertase gives rise to a C5convertase. C3 and C5 convertases of this pathway are regulated by hostcell molecules CR1, DAF, MCP, CD59, and fH. The mode of action of theseproteins involves either decay accelerating activity (i.e., ability todissociate convertases), ability to serve as cofactors in thedegradation of C3b or C4b by factor I, or both. Normally the presence ofcomplement regulatory proteins on host cell surfaces preventssignificant complement activation from occurring thereon.

The C5 convertases produced in both pathways cleave C5 to produce C5aand C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The MAC inserts itself into target cell membranes and causes cell lysis.Small amounts of MAC on the membrane of cells may have a variety ofconsequences other than cell death.

The lectin complement pathway is initiated by binding of mannose-bindinglectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates.The MB 1-1 gene (known as LMAN-1 in humans) encodes a type I integralmembrane protein localized in the intermediate region between theendoplasmic reticulum and the Golgi. The MBL-2 gene encodes the solublemannose-binding protein found in serum. In the human lectin pathway,MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leadingto a C3 convertase described above.

Complement activity is regulated by various mammalian proteins referredto as complement control proteins (CCPs) or regulators of complementactivation (RCA) proteins (U.S. Pat. No. 6,897,290). These proteinsdiffer with respect to ligand specificity and mechanism(s) of complementinhibition. They may accelerate the normal decay of convertases and/orfunction as cofactors for factor I, to enzymatically cleave C3b and/orC4b into smaller fragments. CCPs are characterized by the presence ofmultiple (typically 4-56) homologous motifs known as short consensusrepeats (SCR), complement control protein (CCP) modules, or SUSHIdomains, about 50-70 amino acids in length that contain a conservedmotif including four disulfide-bonded cysteines (two disulfide bonds),proline, tryptophan, and many hydrophobic residues. The CCP familyincludes complement receptor type 1 (CR1; C3b:C4b receptor), complementreceptor type 2 (CR2), membrane cofactor protein (MCP; CD46),decay-accelerating factor (DAF), complement factor H (fH), andC4b-binding protein (C4 bp). CD59 is a membrane-bound complementregulatory protein unrelated structurally to the CCPs. Complementregulatory proteins normally serve to limit complement activation thatmight otherwise occur on cells and tissues of the mammalian, e.g., humanhost. Thus, “self cells are normally protected from the deleteriouseffects that would otherwise ensue were complement activation to proceedon these cells. Deficiencies or defects in complement regulatoryprotein(s) are involved in the pathogenesis of a variety ofcomplement-mediated disorders.

IV. Compstatin Analogs

Compstatin is a cyclic peptide that binds to C3 and inhibits complementactivation. U.S. Pat. No. 6,319,897 describes a peptide having thesequence Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr (SEQ IDNO: 1), with the disulfide bond between the two cysteines denoted bybrackets. It will be understood that the name “compstatin” was not usedin U.S. Pat. No. 6,319,897 but was subsequently adopted in thescientific and patent literature (see, e.g., Morikis, et al, ProteinSci., 7(3):619-27, 1998) to refer to a peptide having the same sequenceas SEQ ID NO: 2 disclosed in U.S. Pat. No. 6,319,897, but amidated atthe C terminus as shown in Table 1 (SEQ ID NO: 8). The term “compstatin”is used herein consistently with such usage (i.e., to refer to SEQ IDNO: 8). Compstatin analogs that have higher complement inhibitingactivity than compstatin have been developed. See, e.g., WO2004/026328(PCT/US2003/029653), Morikis, D., et al, Biochem Soc Trans. 32(Pt1):28-32, 2004, Mallik, B., et al, J. Med. Chem., 274-286, 2005;Katragadda, M., et al. J. Med. Chem., 49: 4616-4622, 2006; WO2007062249(PCT/US20061045539); WO2007044668 (PCT/US2006/039397), WO/2009/046198(PCT/US2008/078593); WO/2010/127336 (PCT/US2010/033345) and discussionbelow.

Compstatin analogs may be acetylated or amidated, e.g., at theN-terminus and/or C-terminus. For example, compstatin analogs may beacetylated at the N-terminus and amidated at the C-terminus. Consistentwith usage in the art, “compstatin” as used herein, and the activitiesof compstatin analogs described herein relative to that of compstatin,refer to compstatin amidated at the C-terminus (Mallik, 2005, supra).

Concatamers or multimers of compstatin or a complement inhibiting analogthereof are also of use in the present invention.

As used herein, the term “compstatin analog” includes compstatin and anycomplement inhibiting analog thereof. The term “compstatin analog”encompasses compstatin and other compounds designed or identified basedon compstatin and whose complement inhibiting activity is at least 50%as great as that of compstatin as measured, e.g., using any complementactivation assay accepted in the art or substantially similar orequivalent assays. Certain suitable assays are described in U.S. Pat.No. 6,319,897, WO2004/026328, Morikis, supra, Mallik, supra, Katragadda2006, supra, WO2007062249 (PCT/US2006/045539); WO2007044668(PCT/US2006/039397), WO/2009/046198 (PCT/US2008/078593); and/orWO/2010/127336 (PCT/US2010/033345). The assay may, for example, measurealternative or classical pathway-mediated erythrocyte lysis or be anELISA assay. In some embodiments, an assay described in WO/2010/135717(PCT/US2010/035871) is used.

The activity of a compstatin analog may be expressed in terms of itsIC₅₀ (the concentration of the compound that inhibits complementactivation by 50%), with a lower IC5₀ indicating a higher activity asrecognized in the art. The activity of a preferred compstatin analog foruse in the present invention is at least as great as that of compstatin.It is noted that certain modifications known to reduce or eliminatecomplement inhibiting activity and may be explicitly excluded from anyembodiment of the invention. The IC5₀ of compstatin has been measured as12 μM using an alternative pathway-mediated erythrocyte lysis assay(WO2004/026328). It will be appreciated that the precise IC5₀ valuemeasured for a given compstatin analog will vary with experimentalconditions (e.g., the serum concentration used in the assay).Comparative values, e.g., obtained from experiments in which IC5₀ isdetermined for multiple different compounds under substantiallyidentical conditions, are of use. In one embodiment, the IC5₀ of thecompstatin analog is no more than the IC5₀ of compstatin. In certainembodiments of the invention the activity of the compstatin analog isbetween 2 and 99 times that of compstatin (i.e., the analog has an ICsothat is less than the IC5₀ of compstatin by a factor of between 2 and99). For example, the activity may be between 10 and 50 times as greatas that of compstatin, or between 50 and 99 times as great as that ofcompstatin. In certain embodiments of the invention the activity of thecompstatin analog is between 99 and 264 times that of compstatin. Forexample, the activity may be 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, or 264 times as great asthat of compstatin. In certain embodiments the activity is between 250and 300, 300 and 350, 350 and 400, or 400 and 500 times as great as thatof compstatin. The invention further contemplates compstatin analogshaving activities between 500 and 1000 times that of compstatin, ormore. In certain embodiments the IC5₀ of the compstatin analog isbetween about 0.2 μM and about 0.5 μM. In certain embodiments the IC5₀of the compstatin analog is between about 0.1 μM and about 0.2 μM. Incertain embodiments the IC5₀ of the compstatin analog is between about0.05 μM and about 0.1 μM. In certain embodiments the IC5₀ of thecompstatin analog is between about 0.001 μM and about 0.05 NM.

¾ e ¾ of compstatin binding to C3 can be measured using isothermaltitration calorimetry (Katragadda, et al, J. Biol. Chem., 279(53),54987-54995, 2004). Binding affinity of a variety of compstatin analogsfor C3 has been correlated with their activity, with a lower Mindicating a higher binding affinity, as recognized in the art. A linearcorrelation between binding affinity and activity was shown for certainanalogs tested (Katragadda, 2004, supra; Katragadda 2006, supra). Incertain embodiments of the invention the compstatin analog binds to C3with a % of between 0.1 μM and 1.0 μM, between 0.05 μM and 0.1 μM,between 0.025 μM and 0.05 μM, between 0.015 μM and 0.025 μM, between0.01 μM and 0.015 μM, or between 0.001 μM and 0.01 μM.

Compounds “designed or identified based on compstatin” include, but arenot limited to, compounds that comprise an amino acid chain whosesequence is obtained by (i) modifying the sequence of compstatin (e.g.,replacing one or more amino acids of the sequence of compstatin with adifferent amino acid or amino acid analog, inserting one or more aminoacids or amino acid analogs into the sequence of compstatin, or deletingone or more amino acids from the sequence of compstatin); (ii) selectionfrom a phage display peptide library in which one or more amino acids ofcompstatin is randomized, and optionally further modified according tomethod (i); or (iii) identified by screening for compounds that competewith compstatin or any analog thereof obtained by methods (i) or (ii)for binding to C3 or a fragment thereof. Many useful compstatin analogscomprise a hydrophobic cluster, a β-turn, and a disulfide bridge.

In certain embodiments of the invention the sequence of the compstatinanalog comprises or consists essentially of a sequence that is obtainedby making 1, 2, 3, or 4 substitutions in the sequence of compstatin,i.e., 1, 2, 3, or 4 amino acids in the sequence of compstatin isreplaced by a different standard amino acid or by a non-standard aminoacid. In certain embodiments of the invention the amino acid at position4 is altered. In certain embodiments of the invention the amino acid atposition 9 is altered. In certain embodiments of the invention the aminoacids at positions 4 and 9 are altered. In certain embodiments of theinvention only the amino acids at positions 4 and 9 are altered. Incertain embodiments of the invention the amino acid at position 4 or 9is altered, or in certain embodiments both amino acids 4 and 9 arealtered, and in addition up to 2 amino acids located at positionsselected from 1, 7, 10, 11, and 13 are altered. In certain embodimentsof the invention the amino acids at positions 4, 7, and 9 are altered.In certain embodiments of the invention amino acids at position 2, 12,or both are altered, provided that the alteration preserves the abilityof the compound to be cyclized. Such alteration(s) at positions 2 and/or12 may be in addition to the alteration(s) at position 1, 4, 7, 9, 10,11. and/or 13. Optionally the sequence of any of the compstatin analogswhose sequence is obtained by replacing one or more amino acids ofcompstatin sequence further includes up to 1, 2, or 3 additional aminoacids at the C-terminus. In one embodiment, the additional amino acid isGly. Optionally the sequence of any of the compstatin analogs whosesequence is obtained by replacing one or more amino acids of compstatinsequence further includes up to 5, or up to 10 additional amino acids atthe C-terminus. It should be understood that compstatin analogs may haveany one or more of the characteristics or features of the variousembodiments described herein, and characteristics or features of anyembodiment may additionally characterize any other embodiment describedherein, unless otherwise stated or evident from the context. In certainembodiments of the invention the sequence of the compstatin analogcomprises or consists essentially of a sequence identical to that ofcompstatin except at positions corresponding to positions 4 and 9 in thesequence of compstatin.

Compstatin and certain compstatin analogs having somewhat greateractivity than compstatin contain only standard amino acids (“standardamino acids” are glycine, leucine, isoleucine, valine, alanine,phenylalanine, tyrosine, tryptophan, aspartic acid, asparagine, glutamicacid, glutamine, cysteine, methionine, arginine, lysine, proline,serine, threonine and histidine). Certain compstatin analogs havingimproved activity incorporate one or more non-standard amino acids.Useful non-standard amino acids include singly and multiply halogenated(e.g., fluorinated) amino acids, D-amino acids, homo-amino acids,N-alkyl amino acids, dehydroamino acids, aromatic amino acids (otherthan phenylalanine, tyrosine and tryptophan), ortho-, meta- orpara-aminobenzoic acid, phospho-amino acids, methoxylated amino acids,and α,α-disubstituted amino acids. In certain embodiments of theinvention, a compstatin analog is designed by replacing one or moreL-amino acids in a compstatin analog described elsewhere herein with thecorresponding D-amino acid. Such compounds and methods of use thereofare an aspect of the invention. Exemplary non-standard amino acids ofuse include 2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI),2-indanylglycine carboxylic acid (2Ig1), dihydrotrpytophan (Dht),4-benzoyl-L-phenylalanine (Bpa), 2-a-aminobutyric acid (2-Abu),3-a-aminobutyric acid (3-Abu), 4-a-aminobutyric acid (4-Abu),cyclohexylalanine (Cha), homocyclohexylalanine (hCha),4-fluoro-L-tryptophan (4fW), 5-fluoro-L-tryptophan (5fW),6-fluoro-L-tryptophan (6fW), 4-hydroxy-L-tryptophan (40H-W),5-hydroxy-L-tryptophan (50H-W), 6-hydroxy-L-tryptophan (60H-W),1-methyl-L-tryptophan (1MeW), 4-methyl-L-tryptophan (4MeW),5-methyl-L-tryptophan (5MeW), 7-aza-L-tryptophan (7aW),a-methyl-L-tryptophan (aMeW), β-methyl-L-tryptophan (PMeW),N-methyl-L-tryptophan (NMeW), ornithine (orn), citrulline, norleucine,γ-glutamic acid, etc.

In certain embodiments of the invention the compstatin analog comprisesone or more Trp analogs (e.g., at position 4 and/or 7 relative to thesequence of compstatin). Exemplary Trp analogs are mentioned above. Seealso Beene, et. al. Biochemistry 41: 10262-10269, 2002 (describing,inter alia, singly- and multiply-halogenated Trp analogs); Babitzke &Yanofsky, J. Biol. Chem. 270: 12452-12456, 1995 (describing, inter alia,methylated and halogenated Trp and other Trp and indole analogs); andU.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and4,299,838. Other Trp analogs include variants that are substituted(e.g., by a methyl group) at the α or β carbon and, optionally, also atone or more positions of the indole ring. Amino acids comprising two ormore aromatic rings, including substituted. unsubstituted, oralternatively substituted variants thereof, are of interest as Trpanalogs. In certain embodiments of the invention the Trp analog, e.g.,at position 4, is 5-methoxy, 5-methyl-, 1-methyl-, or1-formyl-tryptophan. In certain embodiments of the invention a Trpanalog (e.g., at position 4) comprising a 1-alkyl substituent, e.g., alower alkyl (e.g., C₁-C₅) substituent is used. In certain embodiments,N(a) methyl tryptophan or 5-methyltryptophan is used. In someembodiments, an analog comprising a 1-alkanyol substituent, e.g., alower alkanoyl (e.g., C₁-C₅) is used. Examples include1-acetyl-L-tryptophan and L-P-tryptophan.

In certain embodiments the Trp analog has increased hydrophobiccharacter relative to Trp. For example, the indole ring may besubstituted by one or more alkyl (e.g., methyl) groups. In certainembodiments the Trp analog participates in a hydrophobic interactionwith C3. Such a Trp analog may be located. e.g., at position 4 relativeto the sequence of compstatin. In certain embodiments the Trp analogcomprises a substituted or unsubstituted bicyclic aromatic ringcomponent or two or more substituted or unsubstituted monocyclicaromatic ring components.

In certain embodiments the Trp analog has increased propensity to formhydrogen bonds with C3 relative to Trp but does not have increasedhydrophobic character relative to Trp. The Trp analog may have increasedpolarity relative to Trp and/or an increased ability to participate inan electrostatic interaction with a hydrogen bond donor on C3. Certainexemplary Trp analogs with an increased hydrogen bond forming charactercomprise an electronegative substituent on the indole ring. Such a Trpanalog may be located. e.g., at position 7 relative to the sequence ofcompstatin.

In certain embodiments of the invention the compstatin analog comprisesone or more Ala analogs (e.g., at position 9 relative to the sequence ofcompstatin), e.g., Ala analogs that are identical to Ala except thatthey include one or more CH₂ groups in the side chain. In certainembodiments the Ala analog is an unbranched single methyl amino acidsuch as 2-Abu. In certain embodiments of the invention the compstatinanalog comprises one or more Trp analogs (e.g., at position 4 and/or 7relative to the sequence of compstatin) and an Ala analog (e.g., atposition 9 relative to the sequence of compstatin).

In certain embodiments of the invention the compstatin analog is acompound that comprises a peptide that has a sequence of(X′aa)_(n)-Gin-Asp-Xaa-Gly-(X″aa)_(m), (SEQ ID NO: 2) wherein each X′aaand each X″aa is an independently selected amino acid or amino acidanalog, wherein Xaa is Trp or an analog of Trp, and wherein n>1 and m>1and n+m is between 5 and 21. The peptide has a core sequence ofGin-Asp-Xaa-Gly, where Xaa is Trp or an analog of Trp, e.g., an analogof Trp having increased propensity to form hydrogen bonds with an H-bonddonor relative to Trp but, in certain embodiments, not having increasedhydrophobic character relative to Trp. For example, the analog may beone in which the indole ring of Trp is substituted with anelectronegative moiety, e.g., a halogen such as fluorine. In oneembodiment Xaa is 5-fluorotryptophan. Absent evidence to the contrary,one of skill in the art would recognize that any non-naturally occurringpeptide whose sequence comprises this core sequence and that inhibitscomplement activation and/or binds to C3 will have been designed basedon the sequence of compstatin. In an alternative embodiment Xaa is anamino acid or amino acid analog other than a Trp analog that allows theGin-Asp-Xaa-Gly peptide to form a β-turn.

In certain embodiments of the invention the peptide has a core sequenceof X′aa-Gln -Asp-Xaa-Gly (SEQ ID NO: 3), where X′aa and Xaa are selectedfrom Trp and analogs of Trp. In certain embodiments of the invention thepeptide has a core sequence of X′aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3),where X′aa and Xaa are selected from Trp, analogs of Trp, and otheramino acids or amino acid analogs comprising at least one aromatic ring.In certain embodiments of the invention the core sequence forms a β-turnin the context of the peptide. The β-turn may be flexible, allowing thepeptide to assume two or more conformations as assessed for example,using nuclear magnetic resonance (NMR). In certain embodiments X′aa isan analog of Trp that comprises a substituted or unsubstituted bicyclicaromatic ring component or two or more substituted or unsubstitutedmonocyclic aromatic ring components. In certain embodiments of theinvention X′aa is selected from the group consisting of2-napthylalanine, 1-napthylalanine, 2-indanylglycine carboxylic acid,dihydrotryptophan, and benzoylphenylalanine. In certain embodiments ofthe invention X′aa is an analog of Trp that has increased hydrophobiccharacter relative to Trp. For example, X′aa may be 1-methyltryptophan.In certain embodiments of the invention Xaa is an analog of Trp that hasincreased propensity to form hydrogen bonds relative to Trp but, incertain embodiments, not having increased hydrophobic character relativeto Trp. In certain embodiments of the invention the analog of Trp thathas increased propensity to form hydrogen bonds relative to Trpcomprises a modification on the indole ring of Trp, e.g., at position 5,such as a substitution of a halogen atom for an H atom at position 5.For example, Xaa may be 5-fluorotryptophan.

In certain embodiments of the invention the peptide has a core sequenceof X′aa-Gln -Asp-Xaa-Gly-X″aa (SEQ ID NO: 4), where X′aa and Xaa areeach independently selected from Trp and analogs of Trp and X″aa isselected from His, Ala, analogs of Ala, Phe, and Trp. In certainembodiments of the invention X′aa is an analog of Trp that has increasedhydrophobic character relative to Trp, such as 1-methyltryptophan oranother Trp analog having an alkyl substituent on the indole ring (e.g.,at position 1, 4, 5, or 6). In certain embodiments X′aa is an analog ofTrp that comprises a substituted or unsubstituted bicyclic aromatic ringcomponent or two or more substituted or unsubstituted monocyclicaromatic ring components. In certain embodiments of the invention X′aais selected from the group consisting of 2-napthylalanine,1-napthylalanine, 2-indanylglycine carboxylic acid, dihydrotryptophan,and benzoylphenylalanine. In certain embodiments of the invention Xaa isan analog of Trp that has increased propensity to form hydrogen bondswith C3 relative to Trp but, in certain embodiments, not havingincreased hydrophobic character relative to Trp. In certain embodimentsof the invention the analog of Trp that has increased propensity to formhydrogen bonds relative to Trp comprises a modification on the indolering of Trp, e.g., at position 5, such as a substitution of a halogenatom for an H atom at position 5. For example, Xaa may be5-fluorotryptophan. In certain embodiments X″aa is Ala or an analog ofAla such as Abu or another unbranched single methyl amino acid. Incertain embodiments of the invention the peptide has a core sequence ofX′aa-Gln-Asp-Xaa-Gly-X″aa (SEQ ID NO: 4), where X′aa and Xaa are eachindependently selected from Trp, analogs of Trp, and amino acids oramino acid analogs comprising at least one aromatic side chain, and X″aais selected from His, Ala, analogs of Ala, Phe, and Trp. In certainembodiments X″aa is selected from analogs of Trp, aromatic amino acids,and aromatic amino acid analogs.

In certain preferred embodiments of the invention the peptide is cyclic.The peptide may be cyclized via a bond between any two amino acids, oneof which is (X′aa)_(n) and the other of which is located within(X″aa)_(m). In certain embodiments the cyclic portion of the peptide isbetween 9 and 15 amino acids in length, e.g., 10-12 amino acids inlength. In certain embodiments the cyclic portion of the peptide is 11amino acids in length, with a bond (e.g., a disulfide bond) betweenamino acids at positions 2 and 12. For example, the peptide may be 13amino acids long, with a bond between amino acids at positions 2 and 12resulting in a cyclic portion 11 amino acids in length.

In certain embodiments the peptide comprises or consists of the sequenceX′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X′aa1-X″aa2-X″aa3-X″aa4-X″aa5(SEQ ID NO: 5). In certain embodiments X′aa4 and Xaa are selected fromTrp and analogs of Trp, and X′aa1, X′aa2, X′aa3, X′aa1, X″aa2, X″aa3,X″aa4, and X″aa5 are independently selected from among amino acids andamino acid analogs. In certain embodiments X′aa4 and Xaa are selectedfrom aromatic amino acids and aromatic amino acid analogs. Any one ormore of X′aa1, X′aa2, X′aa3, X′aa1, X″aa2, X″aa3, X″aa4, and X″aa5 maybe identical to the amino acid at the corresponding position incompstatin. In one embodiment, X″aa1 is Ala or a single methylunbranched amino acid. The peptide may be cyclized via a covalent bondbetween (i) X′aa1, X′aa2, or X′aa3; and (ii) X″aa2, X″aa3, X″aa4 orX″aa5. In one embodiment the peptide is cyclized via a covalent bondbetween X′aa2 and X″aa4. In one embodiment the covalently bound aminoacid are each Cys and the covalent bond is a disulfide (S—S) bond. Inother embodiments the covalent bond is a C—C, C—O, C—S, or C—N bond. Incertain embodiments one of the covalently bound residues is an aminoacid or amino acid analog having a side chain that comprises a primaryor secondary amine, the other covalently bound residue is an amino acidor amino acid analog having a side chain that comprises a carboxylicacid group, and the covalent bond is an amide bond. Amino acids or aminoacid analogs having a side chain that comprises a primary or secondaryamine include lysine and diaminocarboxylic acids of general structureNH₂(CH₂)nCH(NH₂)COOH such as 2,3-diaminopropionic acid (dapa),2,4-diaminobutyric acid (daba), and ornithine (orn), wherein n=1 (dapa),2 (daba), and 3 (orn), respectively. Examples of amino acids having aside chain that comprises a carboxylic acid group include dicarboxylicamino acids such as glutamic acid and aspartic acid. Analogs such asbeta-hydroxy-L-glutamic acid may also be used. In some embodiments apeptide is cyclized with a thioether bond, e.g., as described inPCT/US2011/052442 (WO/2012/040259). For example, in some embodiments adisulfide bond in any of the peptides is replaced with a thioether bond.In some embodiments, a cystathionine is formed. In some embodiments thecystathionine is a delta-cystathionine or a gamma-cystathionine. In someembodiments a modification comprises replacement of a Cys-Cys disulfidebond between cysteines at X′aa2 and X″aa4 in SEQ ID NO: 5 (orcorresponding positions in other sequences) with addition of a CH₂, toform a homocysteine at X′aa2 or X″aa4, and introduction of a thioetherbond, to form a cystathionine. In one embodiment, the cystathionine is agamma-cystathionine. In another embodiment, the cystathionine is adelta-cystathionine. Another modification in accordance with the presentinvention comprises replacement of the disulfide bond with a thioetherbond without the addition of a CH₂, thereby forming a lantithionine. Insome embodiments a compstatin analog having a thioether in place of adisulfide bond has increased stability, at least under some conditions,as compared with the compstatin analog having the disulfide bond.

In certain embodiments, the compstatin analog is a compound thatcomprises a peptide having a sequence:

(SEQ ID NO: 6) Xaai_C _(ys)-Val-Xaa2-Gin-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4;wherein:Xaa1 is Ile, Val, Leu, E{circumflex over ( )}-Ile, B{circumflex over( )}Val, B¹-Leu or a dipeptide comprising Gly-Ile or B{circumflex over( )}Gly-Ile, and B¹ represents a first blocking moiety;Xaa2 and Xaa2* are independently selected from Trp and analogs of Trp;Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;Xaa4 is L-Thr, D-Thr, He. Val, Gly, a dipeptide selected from Thr-Alaand Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxyterminal —OH of any of the L-Thr, D-Thr, He. Val, Gly, Ala, or Asnoptionally is replaced by a second blocking moiety B²; and the two Cysresidues are joined by a disulfide bond. In some embodiments, Xaa4 isLeu, Nle, His, or Phe or a dipeptide selected from Xaa5-Ala andXaa5-Asn, or a tripeptide Xaa5-Ala-Asn, wherein Xaa5 is selected fromLeu, Nle, His or Phe, and wherein a carboxy terminal —OH of any of theL-Thr, D-Thr, He. Val, Gly, Leu, Nle, His, Phe, Ala, or Asn optionallyis replaced by a second blocking moiety B²; and the two Cys residues arejoined by a disulfide bond.

I, other embodiments Xaa1 is absent or is any amino acid or amino acidanalog, and Xaa2, Xaa2*, Xaa3, and Xaa4 are as defined above. If Xaa1 isabsent, the N-terminal Cys residue may have a blocking moiety B¹attached thereto.

In another embodiment, Xaa4 is any amino acid or amino acid analog andXaa1, Xaa2, Xaa2*, and Xaa3 are as defined above. In another embodimentXaa4 is a dipeptide selected from the group consisting of: Thr-Ala andThr-Asn, wherein the carboxy terminal —OH or the Ala or Asn isoptionally replaced by a second blocking moiety B².

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be Trp.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be an analog of Trp comprising a substituted or unsubstitutedbicyclic aromatic ring component or two or more substituted orunsubstituted monocyclic aromatic ring components. For example, theanalog of Trp may be selected from 2-naphthylalanine (2-Nal),1-naphthylalanine (1-NaI), 2-indanylglycine carboxylic acid (Ig1),dihydrotrpytophan (Dht), and 4-benzoyl-L-phenylalanine.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be an analog of Trp having increased hydrophobic character relativeto Trp. For example, the analog of Trp may be selected from1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan, and6-methyltryptophan. In one embodiment, the analog of Trp is1-methyltryptophan. In one embodiment, Xaa2 is 1-methyltryptophan, Xaa2*is Trp, Xaa3 is Ala, and the other amino acids are identical to those ofcompstatin.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6,Xaa2* may be an analog of Trp such as an analog of Trp having increasedhydrogen bond forming propensity with C3 relative to Trp, which, incertain embodiments, does not have increased hydrophobic characterrelative to Trp. In certain embodiments the analog of Trp comprises anelectronegative substituent on the indole ring. For example, the analogof Trp may be selected from 5-fluorotryptophan and 6-fluorotryptophan.

In certain embodiments of the invention Xaa2 is Trp and Xaa2* is ananalog of Trp having increased hydrogen bond forming propensity with C3relative to Trp which, in certain embodiments, does not have increasedhydrophobic character relative to Trp. In certain embodiments of thecompstatin analog of SEQ ID NO: 6, Xaa2 is analog of Trp havingincreased hydrophobic character relative to Trp such as an analog of Trpselected from 1-methyltryptophan, 4-methyltryptophan,5-methyltryptophan, and 6-methyltryptophan, and Xaa2* is an analog ofTrp having increased hydrogen bond forming propensity with C3 relativeto Trp which, in certain embodiments, does not have increasedhydrophobic character relative to Trp. For example, in one embodimentXaa2 is methyltryptophan and Xaa2* is 5-fluorotryptophan.

In certain of the afore-mentioned embodiments, Xaa3 is Ala. In certainof the afore-mentioned embodiments Xaa3 is a single methyl unbranchedamino acid, e.g., Abu.

The invention further provides compstatin analogs of SEQ ID NO: 6, asdescribed above, wherein Xaa2 and Xaa2* are independently selected fromTrp, analogs of Trp, and other amino acids or amino acid analogs thatcomprise at least one aromatic ring, and Xaa3 is His, Ala or an analogof Ala, Phe, Trp, an analog of Trp, or another aromatic amino acid oraromatic amino acid analog.

In certain embodiments of the invention the blocking moiety present atthe N- or C-terminus of any of the compstatin analogs described hereinis any moiety that stabilizes a peptide against degradation that wouldotherwise occur in mammalian (e.g., human or non-human primate) blood orinterstitial fluid. For example, blocking moiety B¹ could be any moietythat alters the structure of the N-terminus of a peptide so as toinhibit cleavage of a peptide bond between the N-terminal amino acid ofthe peptide and the adjacent amino acid. Blocking moiety B² could be anymoiety that alters the structure of the C-terminus of a peptide so as toinhibit cleavage of a peptide bond between the C-terminal amino acid ofthe peptide and the adjacent amino acid. Any suitable blocking moietiesknown in the art could be used. In certain embodiments of the inventionblocking moiety B¹ comprises an acyl group (i.e., the portion of acarboxylic acid that remains following removal of the —OH group). Theacyl group typically comprises between 1 and 12 carbons, e.g., between 1and 6 carbons. For example, in certain embodiments of the inventionblocking moiety B¹ is selected from the group consisting of: formyl,acetyl, proprionyl, butyryl, isobutyryl, valeryl, isovaleryl, etc. Inone embodiment, the blocking moiety B¹ is an acetyl group, i.e., Xaa1 isAc-He, Ac-Val, Ac-Leu, or Ac-Gly-Ile.

In certain embodiments of the invention blocking moiety B² is a primaryor secondary amine (—NH₂ or —NHR¹, wherein R is an organic moiety suchas an alkyl group).

In certain embodiments of the invention blocking moiety B¹ is any moietythat neutralizes or reduces the positive charge that may otherwise bepresent at the N-terminus at physiological pH. In certain embodiments ofthe invention blocking moiety B² is any moiety that neutralizes orreduces the negative charge that may otherwise be present at theC-terminus at physiological pH.

In certain embodiments of the invention, the compstatin analog isacetylated or amidated at the N-terminus and/or C-terminus,respectively. A compstatin analog may be acetylated at the N-terminus,amidated at the C-terminus, and or both acetylated at the N-terminus andamidated at the C-terminus. In certain embodiments of the invention acompstatin analog comprises an alkyl or aryl group at the N-terminusrather than an acetyl group.

In certain embodiments, the compstatin analog is a compound thatcomprises a peptide having a sequence:

(SEQ ID NO: 7) Xaa1_C_(ys) - Val - Xaa2 - Gin - Asp - Xaa2* - Gly -Xaa3 - His - Arg - Cys - Xaa4;wherein:Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprisingGly-He or Ac-Gly-Ile;Xaa2 and Xaa2* are independently selected from Trp and analogs of Trp;Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Alaand Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxyterminal —OH of any of L-Thr, D-Thr, He. Val, Gly, Ala, or Asnoptionally is replaced by —NH₂; and the two Cys residues are joined by adisulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe or adipeptide selected from Xaa5-Ala and Xaa5-Asn, or a tripeptideXaa5-Ala-Asn, wherein Xaa5 is selected from Leu, Nle, His or Phe, andwherein a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val,Gly, Leu, Nle, His, Phe, Ala, or Asn optionally is replaced by a secondblocking moiety B2; and the two Cys residues are joined by a disulfidebond.

In some embodiments, Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as describedabove for the various embodiments of SEQ ID NO: 6. For example, incertain embodiments Xaa2* is Trp. In certain embodiments Xaa2 is ananalog of Trp having increased hydrophobic character relative to Trp,e.g., 1-methyltryptophan. In certain embodiments Xaa3 is Ala. In certainembodiments Xaa3 is a single methyl unbranched amino acid.

In certain embodiments of the invention Xaa1 is He and Xaa4 is L-Thr.

In certain embodiments of the invention Xaa1 is He. Xaa2* is Trp, andXaa4 is L-Thr.

¾ e invention further provides compstatin analogs of SEQ ID NO: 7, asdescribed above, wherein Xaa2 and Xaa2* are independently selected fromTrp, analogs of Trp, other amino acids or aromatic amino acid analogs,and Xaa3 is His, Ala or an analog of Ala, Phe, Trp, an analog of Trp, oranother aromatic amino acid or aromatic amino acid analog.

In certain embodiments of any of the compstatin analogs describedherein, an analog of Phe is used rather than Phe.

Table 1 provides a non-limiting list of compstatin analogs useful in thepresent invention. The analogs are referred to in abbreviated form inthe left column by indicating specific modifications at designatedpositions (1-13) as compared to the parent peptide, compstatin.Consistent with usage in the art, “compstatin” as used herein, and theactivities of compstatin analogs described herein relative to that ofcompstatin, refer to the compstatin peptide amidated at the C-terminus.Unless otherwise indicated, peptides in Table 1 are amidated at theC-terminus. Bold text is used to indicate certain modifications.Activity relative to compstatin is based on published data and assaysdescribed therein (WO2004/026328, WO2007044668, Mallik, 2005;Katragadda, 2006). Where multiple publications reporting an activitywere consulted, the more recently published value is used, and it willbe recognized that values may be adjusted in the case of differencesbetween assays. It will also be appreciated that in certain embodimentsof the invention the peptides listed in Table 1 are cyclized via adisulfide bond between the two Cys residues when used in the therapeuticcompositions and methods of the invention. Alternate means for cyclizingthe peptides are also within the scope of the invention. As noted above,in various embodiments of the invention one or more amino acid(s) of acompstatin analog (e.g., any of the compstatin analogs disclosed herein)can be an N-alkyl amino acid (e.g., an N-methyl amino acid). Forexample, and without limitation, at least one amino acid within thecyclic portion of the peptide, at least one amino acid N-terminal to thecyclic portion, and/or at least one amino acid C-terminal to the cyclicportion may be an N-alkyl amino acid, e.g., an N-methyl amino acid. Insome embodiments of the invention, for example, a compstatin analogcomprises an N-methyl glycine, e.g., at the position corresponding toposition 8 of compstatin and/or at the position corresponding toposition 13 of compstatin. In some embodiments, one or more of thecompstatin analogs in Table 1 contains at least one N-methyl glycine,e.g., at the position corresponding to position 8 of compstatin and/orat the position corresponding to position 13 of compstatin. In someembodiments, one or more of the compstatin analogs in Table 1 containsat least one N-methyl isoleucine, e.g., at the position corresponding toposition 13 of compstatin. For example, a Thr at or near the C-terminalend of a peptide whose sequence is listed in Table 1 or any othercompstatin analog sequence may be replaced by N-methyl Ile. As will beappreciated, in some embodiments the N-methylated amino acids compriseN-methyl Gly at position 8 and N-methyl Ile at position 13. In someembodiments the N-methylated amino acids comprise N-methyl Gly in a coresequence such as SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments theN-methylated amino acids comprise N-methyl Gly in a core sequence suchas SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

TABLE 1 SEQ ID Activity over Peptide Sequence NO: compstatin CompstatinH-ICVVQDWGHHRCT-CONH2 8 * Ac-compstatin AC-ICVVQDWGHHRCT-CONH2 9 3xmoreAc-V4Y/H9A Ac-ICV Y QDWG A HRCT-CONH2 10 14xmore Ac-V4W/H9A -OH Ac-ICV WQDWG A HRCT-COOH 11 27xmore AC-V4W/H9A AC-ICV W QDWG A HRCT-CONH2 1245xmore AC-V4W/H9A/T13dT -OH Ac-ICV W QDWG A HRC dT -COOH 13 55xmoreAc-V4(2-Nal)/H9A Ac-ICV (2-Nal) QDWG A HRCT-CONH2 14 99xmoreAc V4(2-Nal)/H9A -OH Ac-ICV (2-Nal) QDWG A HRCT-COOH 15 38xmoreAc V4(1-Nal)/H9A -OH Ac-ICV (1-Nal) QDWG A HRCT-COOH 16 30xmoreAc-V42lgl/H9A Ac-ICV (2-Igl) QDWG A HRCT-CONH2 17 39xmoreAc-V42lgl/H9A -OH Ac-ICV (2-Igl) QDWG A HRCT-COOH 18 37xmoreAc-V4Dht/H9A -OH Ac-ICV Dht QDWG A HRCT-COOH 19 5xmoreAc-V4(Bpa)/H9A -OH Ac-ICV (Bpa) QDWG A HRCT-COOH 20 49xmoreAc-V4(Bpa)/H9A Ac-ICV (Bpa) QDWG A HRCT-CONH2 21 86xmoreAc-V4(Bta)/H9A -OH Ac-ICV (Bta) QDWG A HRCT-COOH 22 65xmoreAc-V4(Bta)/H9A Ac-ICV (Bta) QDWG A HRCT-CONH2 23 64xmoreAc-V4W/H9(2-Abu) Ac-ICV W QDWG(2- Abu) HRCT-CONH2 24 64xmore+G/V4W/H9A +AN -OH H- G ICV W QDWG A HRCTA N -COOH 25 38xmoreAc-V4(5fW)/H9A Ac-ICV (5fW) QDWG A HRCT- CONH₂ 26 31xmoreAc-V4(5-MeW)/H9A Ac-ICV (5-methyl-W) QDWG A HRCT- CONH₂ 27 67xmoreAc-V4(1-MeW)/H9A Ac-ICV (1-methyl-W) QDWG A HRCT- CONH₂ 28 264xmoreAc-V4W/W7(5fW)/H9A Ac-ICV W QD (5fW) G A HRCT-CONH₂ 29 121xmoreAc-V4(5fW)/W7(5fW)/H9A Ac-ICV (5fW) QD (5fW) G A HRCT- CONH₂ 30 NAAc-V4(5-MeW)/W7(5fW)H9A Ac-ICV (5-methyl-W) QD (5fW) G A HRCT- 31 NACONH₂ Ac-V4(1MeW)/W7(5fW)/H9A Ac-ICV(1-methyl-W)QD (5fW) G A HRCT- 32264xmore CONH₂ +G/V4(6fW)/W7(6fW)H9A+N- H-GICV (6fW )QD(6fW)G A HRCT N-COOH 33 126xmore OH Ac-V4(1-formyl-W)/H9A Ac-ICV( 1-formyl-W )QDWG AHRCT-CONH₂ 34 264xmore Ac-V4(5-methoxy-W)/H9A Ac-ICV (1-methyoxy-W )QDWGA HRCT- 35 76xmore CONH₂ G/V4(5f-W)/W7(5fW)/H9A+N- H-GICV( 5fW )QD( 5fW)G A HRCT N -COOH 36 112xmore OH NA = not available

In certain embodiments of the compositions and methods of the inventionthe compstatin analog has a sequence selected from sequences 9-36. Incertain embodiments of the compositions and methods of the invention thecompstatin analog has a sequence selected from SEQ ID NOs: 14, 21, 28,29, 32, 33, 34, and 36. In certain embodiments of the compositionsand/or methods of the invention the compstatin analog has a sequenceselected from SEQ ID NOs: 30 and 31. In one embodiment of thecompositions and methods of the invention the compstatin analog has asequence of SEQ ID NO: 28. In one embodiment of the compositions andmethods of the invention the compstatin analog has a sequence of SEQ IDNO: 32. In one embodiment of the compositions and methods of theinvention the compstatin analog has a sequence of SEQ ID NO: 34. In oneembodiment of the compositions and methods of the invention thecompstatin analog has a sequence of SEQ ID NO: 36.

In some embodiments a blocking moiety B¹ comprises an amino acid, whichmay be represented as XaaO. In some embodiments blocking moiety B²comprises an amino acid, which may be represented as XaaN. In someembodiments blocking moiety B¹ and/or B² comprises a non-standard aminoacid, such as a D-amino acid, N-alkyl amino acid (e.g., N-methyl aminoacid). In some embodiments a blocking moiety B¹ and/or B² comprises anon-standard amino acid that is an analog of a standard amino acid. Insome embodiments an amino acid analog comprises a lower alkyl, loweralkoxy, or halogen substituent, as compared with a standard amino acidof which it is an analog. In some embodiments a substituent is on a sidechain. In some embodiments a substituent is on an alpha carbon atom. Insome embodiments, a blocking moiety B¹ comprising an amino acid, e.g., anon-standard amino acid, further comprises a moiety B^(1a). For example,blocking moiety B¹ may be represented as B^(1a)-Xaa0. In someembodiments Bia neutralizes or reduces a positive charge that mayotherwise be present at the N-terminus at physiological pH. In someembodiments B^(1a) comprises or consists of, e.g., an acyl group that,e.g., comprises between 1 and 12 carbons, e.g., between 1 and 6 carbons.In certain embodiments blocking moiety B^(1a) is selected from the groupconsisting of: formyl, acetyl, proprionyl, butyryl, isobutyryl, valeryl,isovaleryl, etc. In some embodiments, a blocking moiety B² comprising anamino acid, e.g., a non-standard amino acid, may further comprise amoiety B^(2a) For example, blocking moiety B² may be represented asXaaN-B^(2a), where N represents the appropriate number for the aminoacid (which will depend on the numbering used in the rest of thepeptide). In some embodiments B^(2a) neutralizes or reduces a negativecharge that may otherwise be present at the C-terminus at physiologicalpH. In some embodiments B^(2a) comprises or consists of a primary orsecondary amine (e.g., NH₂). It will be understood that a blockingactivity of moiety B^(1a)-Xaa0 and/or XaaN-B^(2a) may be provided byeither or both components of the moiety in various embodiments. In someembodiments a blocking moiety or portion thereof, e.g., an amino acidresidue, may contribute to increasing affinity of the compound for C3 orC3b and/or improve the activity of the compound. In some embodiments acontribution to affinity or activity of an amino acid residue may be atleast as important as a contribution to blocking activity. For example,in some embodiments XaaO and/or XaaN in B^(1a)-Xaa0 and/or XaaN-B^(2a)may function mainly to increase affinity or activity of the compound,while B^(1a) and/or B^(2a) may inhibit digestion of and/or neutralize acharge of the peptide. In some embodiments a compstatin analog comprisesthe amino acid sequence of any of SEQ ID NOs: 5-36, wherein SEQ ID NOs:5-36 is further extended at the N- and/or C-terminus. In someembodiments, the sequence may be represented asB^(1a)-Xaa0-SEQUENCE-XaaN-B^(2a), where SEQUENCE represents any of SEQID NOs: 5-36, wherein Bia and B^(2a) may independently be present orabsent. For example, in some embodiments a compstatin analog comprisesB^(1a)-Xaa0-X′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X″aa1-X″aa2-X″aa3-X″aa4-X″aa5-XaaN-B²^(a) (SEQ ID NO: 37A), where X′aa1-X′aa2-X′aa3-X′aa4, Xaa, X″aa1, X″aa2,X″aa3, X″aa4, and X″aa5 are as set forth above for SEQ ID NO: 5.

In some embodiments a compstatin analog comprisesB^(1a)-Xaa0-Xaa1-Cys-Val-Xaa2-Gin-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4-XaaN-B²^(a) (SEQ ID NO: 38A), where Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are asset forth above for SEQ ID NO: 6 or wherein Xaa1, Xaa2, Xaa2*, Xaa3, andXaa4 are as set forth for SEQ ID NO: 6 or SEQ ID NO: 7.

In some embodiments a compstatin analog comprisesB^(1a)-Xaa0-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-XaaN-B² ^(a) (SEQ ID NO: 39A) wherein Xaa1, Xaa2,Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa1O, Xaa1l, Xaa12, and Xaa13are identical to amino acids at positions 1-13 of any of SEQ ID NOs:9-36.

In some embodiments XaaO and/or XaaN in any compstatin analog sequencecomprises an amino acid that comprises an aromatic ring having an alkylsubstituent at one or more positions. In some embodiments an alkylsubstituent is a lower alkyl substituent. For example, in someembodiments an alkyl substituent is a methyl or ethyl group. In someembodiments a substituent is located at any position that does notdestroy the aromatic character of the compound. In some embodiments asubstituent is located at any position that does not destroy thearomatic character of a ring to which the substituent is attached. Insome embodiments a substituent is located at position 1, 2, 3, 4, or 5.In some embodiments XaaO comprises an O-methyl analog of tyrosine,2-hydroxyphenylalanine or 3-hydroxyphenylalanine. For purposes of thepresent disclosure, a lower case “m” followed by a three letter aminoacid abbreviation may be used to specifically indicate that the aminoacid is an N-methyl amino acid. For example, where the abbreviation“mGly” appears herein, it denotes N-methyl glycine (also sometimesreferred to as sarcosine or Sar). In some embodiments XaaO is orcomprises mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), Cha, mPhe, mVal, mile,mAla, DTyr, DPhe, DArg, DTrp, DThr, DTyr(Me), mPhe, mVal, mile, DAla, orDCha. For example, in some embodiments a compstatin analog comprises apeptide having a sequenceB¹-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B² (SEQID NO: 40A) orB¹-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B² (SEQID NO: 41A). The two Cys residues are joined by a disulfide bond in theactive compounds. In some embodiments the peptide is acetylated at theN-terminus and/or amidated at the C-terminus. In some embodiments B¹comprises B^(1a)-Xaa0 and/or B² comprises XaaN-B^(2a), as describedabove. For example, in some embodiments B¹ comprises or consists of Gly,mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), mPhe, mVal, mile, mAla, DTyr,DPhe, DTrp, DCha, DAla and B² comprises NH₂, e.g., a carboxy terminal—OH of mile is replaced by NH₂. In some embodiments B¹ comprises orconsists of mGly, Tyr, DTyr, or Tyr(Me) and B² comprises NH₂, e.g., acarboxy terminal —OH of mile is replaced by N¾. In some embodiments anIle at position Xaa1 is replaced by Gly. Complement inhibition potencyand/or C3b binding parameters of selected compstatin analogs aredescribed in WO/2010/127336 (PCT/US2010/033345) and/or in Qu, et al,Immunobiology (2012), doi:10.1016/j.imbio.2012.06.003.

In some embodiments a blocking moiety or portion thereof, e.g., an aminoacid residue, may contribute to increasing affinity of the compound forC3 or C3b and/or improve the activity of the compound. In someembodiments a contribution to affinity or activity of an amino acid oramino acid analog may be more significant than a blocking activity.

In certain embodiments of the compositions and methods of the inventionthe compstatin analog has a sequence as set forth in Table 1, but wherethe Ac-group is replaced by an alternate blocking moiety B¹, asdescribed herein. In some embodiments the —N¾ group is replaced by analternate blocking moiety B², as described herein.

In one embodiment, the compstatin analog binds to substantially the sameregion of the β chain of human C3 as does compstatin. In one embodimentthe compstatin analog is a compound that binds to a fragment of theC-terminal portion of the β chain of human C3 having a molecular weightof about 40 kDa to which compstatin binds (Soulika, A. M., et al, Mol.Immunol, 35:160, 1998; Soulika, A. M., et al., Mol. Immunol.43(12):2023-9, 2006). In certain embodiments the compstatin analog is acompound that binds to the binding site of compstatin as determined in acompstatin-C3 structure, e.g., a crystal structure or NMR-derived 3Dstructure. In certain embodiments the compstatin analog is a compoundthat could substitute for compstatin in a compstatin-C3 structure andwould form substantially the same intermolecular contacts with C3 ascompstatin. In certain embodiments the compstatin analog is a compoundthat binds to the binding site of a peptide having a sequence set forthin Table 1, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, 37, 37A,38A, 39A, 40A, or 41A or another compstatin analog sequence disclosedherein in a peptide-C3 structure, e.g., a crystal structure. In certainembodiments the compstatin analog is a compound that binds to thebinding site of a peptide having SEQ ID NO: 30 or 31 in a peptide-C3structure, e.g., a crystal structure. In certain embodiments thecompstatin analog is a compound that could substitute for the peptide ofSEQ ID NO: 9-36, e.g., a compound that could substitute for the peptideof SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, 37, 37A, 38A, 39A, 40A,or 41A or another compstatin analog sequence disclosed herein in apeptide-C3 structure and would form substantially the sameintermolecular contacts with C3 as the peptide. In certain embodimentsthe compstatin analog is a compound that could substitute for thepeptide of SEQ ID NO: 30 or 31 in a peptide-C3 structure and would formsubstantially the same intermolecular contacts with C3 as the peptide.

Q_(ne) of ordinary skill in the art will readily be able to determinewhether a compstatin analog binds to a fragment of the C-terminalportion of the β chain of C3 using routine experimental methods. Forexample, one of skill in the art could synthesize a photocrosslinkableversion of the compstatin analog by including a photo-crosslinking aminoacid such as p-benzoyl-L-phenylalanine (Bpa) in the compound, e.g., atthe C-terminus of the sequence (Soulika, A. M., et al, supra).Optionally additional amino acids, e.g., an epitope tag such as a FLAGtag or an HA tag could be included to facilitate detection of thecompound, e.g., by Western blotting. The compstatin analog is incubatedwith the fragment and crosslinking is initiated. Colocalization of thecompstatin analog and the C3 fragment indicates binding. Surface plasmonresonance may also be used to determine whether a compstatin analogbinds to the compstatin binding site on C3 or a fragment thereof. One ofskill in the art would be able to use molecular modeling softwareprograms to predict whether a compound would form substantially the sameintermolecular contacts with C3 as would compstatin or a peptide havingthe sequence of any of the peptides in Table 1, e.g., SEQ ID NO: 14, 21,28, 29, 32, 33, 34, or 36, or in some embodiments SEQ ID NO: 30 or 31,37, 37A, 38A, 39A, 40A, or 41A or another compstatin analog sequencedisclosed herein.

Compstatin analogs may be prepared by various synthetic methods ofpeptide synthesis known in the art via condensation of amino acidresidues, e.g., in accordance with conventional peptide synthesismethods, may be prepared by expression in vitro or in living cells fromappropriate nucleic acid sequences encoding them using methods known inthe art. For example, peptides may be synthesized using standardsolid-phase methodologies as described in Malik, supra, Katragadda,supra, WO2004026328, and/or WO2007062249. Potentially reactive moietiessuch as amino and carboxyl groups, reactive functional groups, etc., maybe protected and subsequently deprotected using various protectinggroups and methodologies known in the art. See, e.g., “Protective Groupsin Organic Synthesis”, 3^(rd) ed. Greene, T. W. and Wuts, P. G., Eds.,John Wiley & Sons, New York: 1999. Peptides may be purified usingstandard approaches such as reversed-phase HPLC. Separation ofdiasteriomeric peptides, if desired, may be performed using knownmethods such as reversed-phase HPLC. Preparations may be lyophilized, ifdesired, and subsequently dissolved in a suitable solvent, e.g., water.The pH of the resulting solution may be adjusted, e.g. to physiologicalpH, using a base such as NaOH. Peptide preparations may be characterizedby mass spectrometry if desired, e.g., to confirm mass and/or disulfidebond formation. See, e.g., Mallik, 2005, and Katragadda, 2006.

A compstatin analog, optionally linked to a cell penetrating moiety, canbe modified by addition of a molecule such as polyethylene glycol (PEG)or similar molecules to stabilize the compound, reduce itsimmunogenicity, increase its lifetime in the body, increase or decreaseits solubility, and/or increase its resistance to degradation. Methodsfor pegylation are well known in the art (Veronese, F. M. & Harris, Adv.Drug Deliv. Rev. 54, 453-456, 2002; Davis, F. F., Adv. Drug Deliv. Rev.54, 457-458, 2002); Hinds, K. D. & Kim, S. W. Adv. Drug Deliv. Rev. 54,505-530 (2002; Roberts, M. J., Bentley, M. D. & Harris, J. M. Adv. DrugDeliv. Rev. 54, 459-476; 2002); Wang, Y. S. et al. Adv. Drug Deliv. Rev.54, 547-570, 2002). A wide variety of polymers such as PEGs and modifiedPEGs, including derivatized PEGs to which polypeptides can convenientlybe attached are described in Nektar Advanced Pegylation 2005-2006Product Catalog, Nektar Therapeutics, San Carlos, Calif., which alsoprovides details of appropriate conjugation procedures. In someembodiments a compstatin analog is fused to the Fc domain of animmunoglobulin or a portion thereof. In some other embodiments acompstatin analog is conjugated to an albumin moiety or to an albuminbinding peptide. Thus in some embodiments a CPCA is modified with one ormore polypeptide or non-polypeptide components, e.g., the compstatinanalog is pegylated or conjugated to another moiety. In some embodimentsthe component is not the Fc domain of an immunoglobulin or a portionthereof. A compstatin analog can be provided as a multimer or as part ofa supramolecular complex, which can include either a single molecularspecies or multiple different species (e.g., multiple differentanalogs).

In some embodiments, a compstatin analog is a multivalent compoundcomprising a plurality of cell-penetrating compstatin analog moietiescovalently or noncovalently linked to a polymeric backbone or scaffold.The cell-penetrating compstatin analog moieties can be identical ordifferent. In certain embodiments of the invention the multivalentcompound comprises multiple instances, or copies, of a singlecell-penetrating compstatin analog moiety. In other embodiments of theinvention the multivalent compound comprises one or more instances ofeach of two of more non-identical cell-penetrating compstatin analogmoieties, e.g., 3, 4, 5, or more different cell-penetrating compstatinanalog moieties. In certain embodiments of the invention the number ofcell-penetrating compstatin analog moieties (“n”) is between 2 and 6. Inother embodiments of the invention n is between 7 and 20. In otherembodiments of the invention n is between 20 and 100. In otherembodiments n is between 100 and 1,000. In other embodiments of theinvention n is between 1,000 and 10,000. In other embodiments n isbetween 10,000 and 50,000. In other embodiments n is between 50,000 and100,000. In other embodiments n is between 100,000 and 1,000,000.

The cell-penetrating compstatin analog moieties may be attached directlyto the polymeric scaffold or may be attached via a linking moiety thatconnects the compstatin analog moiety to the polymeric scaffold. Thelinking moiety may be attached to a single cell-penetrating compstatinanalog moiety and to the polymeric scaffold. Alternately, a linkingmoiety may have multiple cell-penetrating compstatin analog moietiesjoined thereto so that the linking moiety attaches multiplecell-penetrating compstatin analog moieties to the polymeric scaffold.In some embodiments the polymeric scaffold may further have at leastsome compstatin analog moieties that do not comprise a CPM attachedthereto. In some embodiments at least some compstatin analog moieties,e.g., cell-penetrating compstatin analog moieties, are attached via acleavable linker. The linker may be hydrolysable or susceptible toenzyme-mediated cleavage in the blood or extravascular space, whereinsuch cleavage may release active compstatin analogs, e.g.,cell-penetrating compstatin analogs. Released cell-penetratingcompstatin analogs may be internalized by cells, e.g., at or near theirsite of release or may be carried in the circulatory system to beinternalized by cells at distant locations or in the blood or lymph.

In some embodiments, the compstatin analog comprises an amino acidhaving a side chain comprising a primary or secondary amine, e.g., a Lysresidue. For example, a Lys residue, or a sequence comprising a Lysresidue, is added at the N-terminus and/or C-terminus of the compstatinanalog. In some embodiments, the Lys residue is separated from thecyclic portion of the compstatin analog by a rigid or flexible spacer.The spacer may, for example, comprise a substituted or unsubstituted,saturated or unsaturated alkyl chain, oligo(ethylene glycol) chain,and/or other moieties, e.g., as described in Section VI with regard tolinkers. The length of the chain may be, e.g., between 2 and 20 carbonatoms. In other embodiments the spacer is a peptide. The peptide spacermay be, e.g., between 1 and 20 amino acids in length, e.g., between 4and 20 amino acids in length. Suitable spacers can comprise or consistof multiple Gly residues, Ser residues, or both, for example.Optionally, the amino acid having a side chain comprising a primary orsecondary amine and/or at least one amino acid in a spacer is a D-aminoacid. Any of a variety of polymeric backbones or scaffolds could beused. For example, the polymeric backbone or scaffold may be apolyamide, polysaccharide, polyanhydride, polyacrylamide,polymethacrylate, polypeptide, polyethylene oxide, or dendrimer.Suitable methods and polymeric backbones are described, e.g., inWO98/46270 (PCT/US98/07171) or WO98/47002 (PCT/US98/06963). In oneembodiment, the polymeric backbone or scaffold comprises multiplereactive functional groups, such as carboxylic acids, anhydride, orsuccinimide groups. The polymeric backbone or scaffold is reacted withthe compstatin analogs. In one embodiment, the compstatin analogcomprises any of a number of different reactive functional groups, suchas carboxylic acids, anhydride, or succinimide groups, which are reactedwith appropriate groups on the polymeric backbone. Alternately,monomeric units that could be joined to one another to form a polymericbackbone or scaffold are first reacted with the compstatin analogs andthe resulting monomers are polymerized. In another embodiment, shortchains are prepolymerized, functionalized, and then a mixture of shortchains of different composition are assembled into longer polymers.

V. Compstatin Mimetics

The structure of compstatin is known in the art, and NMR structures fora number of compstatin analogs having higher activity than compstatinare also known (Malik, supra). Structural information may be used todesign compstatin mimetics.

In one embodiment, the compstatin mimetic is any compound that competeswith compstatin or any compstatin analog (e.g., a compstatin analogwhose sequence is set forth in Table 1) for binding to C3 or a fragmentthereof (such as a 40 kD fragment of the β chain to which compstatinbinds). In some embodiments, the compstatin mimetic has an activityequal to or greater than that of compstatin. In some embodiments, thecompstatin mimetic is more stable, orally available, or has a betterbioavailability than compstatin. The compstatin mimetic may be apeptide, nucleic acid, or small molecule. In certain embodiments thecompstatin mimetic is a compound that binds to the binding site ofcompstatin as determined in a compstatin-C3 structure, e.g., a crystalstructure or a 3-D structure derived from NMR experiments. In certainembodiments the compstatin mimetic is a compound that could substitutefor compstatin in a compstatin-C3 structure and would form substantiallythe same intermolecular contacts with C3 as compstatin. In certainembodiments the compstatin mimetic is a compound that binds to thebinding site of a peptide having a sequence set forth in Table 1, e.g.,SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or in certain embodimentsSEQ ID NO: 30 or 31 or other compstatin analog sequence, in a peptide-C3structure. In certain embodiments the compstatin mimetic is a compoundthat could substitute for a peptide having a sequence set forth in Table1, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or in certainembodiments SEQ ID NO: 30 or 31 or other compstatin analog sequence, ina peptide-C3 structure and would form substantially the sameintermolecular contacts with C3 as the peptide. In certain embodimentsthe compstatin mimetic has a non-peptide backbone but has side chainsarranged in a sequence designed based on the sequence of compstatin.

One of skill in the art will appreciate that once a particular desiredconformation of a short peptide has been ascertained, methods fordesigning a peptide or peptidomimetic to fit that conformation are wellknown. See, e.g., G. R. Marshall (1993), Tetrahedron, 49: 3547-3558;Hruby and Nikiforovich (1991), in Molecular Conformation and BiologicalInteractions, P. Balaram & S. Ramasehan, eds., Indian Acad. of Sci.,Bangalore, P P. 429-455), Eguchi M, Kahn M., Mini Rev Med Chem.,2(5):447-62, 2002. Of particular relevance to the present invention, thedesign of peptide analogs may be further refined by considering thecontribution of various side chains of amino acid residues, e.g., forthe effect of functional groups or for steric considerations asdescribed in the art for compstatin and analogs thereof, among others.

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for the purpose of providing thespecific backbone conformation and side chain functionalities requiredfor binding to C3 and inhibiting complement activation. Accordingly, itis contemplated as being within the scope of the present invention toproduce and utilize C3-binding, complement-inhibiting compounds throughthe use of either naturally-occurring amino acids, amino acidderivatives, analogs or non-amino acid molecules capable of being joinedto form the appropriate backbone conformation. A non-peptide analog, oran analog comprising peptide and non-peptide components, is sometimesreferred to herein as a “peptidomimetic” or “isosteric mimetic,” todesignate substitutions or derivations of a peptide that possesses muchthe same backbone conformational features and/or other functionalities,so as to be sufficiently similar to the exemplified peptides to inhibitcomplement activation. More generally, a compstatin mimetic is anycompound that would position pharmacophores similarly to theirpositioning in compstatin, even if the backbone differs.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art. Assuming rotational constraintssimilar to those of amino acid residues within a peptide, analogscomprising non-amino acid moieties may be analyzed, and theirconformational motifs verified, by means of the Ramachandran plot (Hruby& Nikiforovich 1991), among other known techniques.

One of skill in the art will readily be able to establish suitablescreening assays to identify additional compstatin mimetics and toselect those having desired inhibitory activities. For example,compstatin or an analog thereof could be labeled (e.g., with aradioactive or fluorescent label) and contacted with C3 in the presenceof different concentrations of a test compound. The ability of the testcompound to diminish binding of the compstatin analog to C3 isevaluated. A test compound that significantly diminishes binding of thecompstatin analog to C3 is a candidate compstatin mimetic. For example,a test compound that diminishes steady-state concentration of acompstatin analog-C3 complex, or that diminishes the rate of formationof a compstatin analog-C3 complex by at least 25%, or by at least 50%,is a candidate compstatin mimetic. One of skill in the art willrecognize that a number of variations of this screening assay may beemployed. Compounds to be screened include natural products, librariesof aptamers, phage display libraries, compound libraries synthesizedusing combinatorial chemistry, etc. The invention encompassessynthesizing a combinatorial library of compounds based upon the coresequence described above and screening the library to identifycompstatin mimetics. Any of these methods could also be used to identifynew compstatin analogs having higher inhibitory activity than compstatinanalogs tested thus far. It will be appreciated that compstatin mimeticscould be used in cell-penetrating compstatin analogs of the invention,and the invention provides such cell-penetrating compstatin analogmimetics.

VI. Cell-Penetrating Compstatin Analogs

The invention provides a variety of cell-penetrating compstatin analogs.In some aspects, a cell-penetrating compstatin analog comprises acompound of formula A-L-M, wherein A is a moiety that comprises a cellpenetrating moiety (CPM), L is an optionally present linking portion,and M comprises a compstatin analog moiety. The compstatin analog moietycan comprise any compstatin analog, e.g., any compstatin analogdescribed above, in various embodiments. Formula A-L-M encompassesembodiments in which A-L is present at the N-terminus of the compstatinanalog moiety, embodiments in which A-L is present at the C-terminus ofthe compstatin analog moiety, embodiments in which A-L is attached to aside chain of an amino acid of the compstatin analog moiety, andembodiments where the same or different A-Ls are present at both ends ofM. It will be appreciated that when certain compstatin analog(s) arepresent as a compstatin analog moiety in a compound of formula A-L-M, afunctional group of the compstatin analog will have reacted with afunctional group of L to form a covalent bond to A or L. For example, acell-penetrating compstatin analog in which the compstatin analog moietycomprises a compstatin analog that contains an amino acid with a sidechain containing a primary amine (NFL) group (which compstatin analogcan be represented by formula R¹-(NFL)), can have a formula R¹—NH-L-A inwhich a new covalent bond to L (e.g., N—C) has been formed and ahydrogen lost. Thus the term “compstatin analog moiety” includesmolecular structures in which at least one atom of a compstatin analogparticipates in a covalent bond with a second moiety, which may, e.g.,modification of a side chain. Similar considerations apply to compstatinanalog moieties present in multivalent compound described above. In someembodiments, a blocking moiety at the N-terminus or C-terminus of acompstatin analog, e.g., a compstatin analog described in Section IVabove, is replaced by A-L in the structure of a cell-penetratingcompstatin analog. In some embodiments, A or L comprises a blockingmoiety. In some embodiments, a cell-penetrating compstatin analog has amolar activity of at least about 10%, 20%, or 30%, e.g., between 30% and40%, between 30% and 50%, between 30% and 60%, between 30% and 70%,between 30% and 80%, between 30% and 90%, or more, of the activity of acorresponding compstatin analog having the same amino acid sequence(and, if applicable, one or more blocking moiet(ies)) but not comprisinga cell penetrating moiety. In some embodiments in which acell-penetrating compstatin analog comprises multiple compstatin analogmoieties, the molar activity of the cell-penetrating compstatin analogis at least about 10%, 20%, or 30%, e.g., between 30% and 40%, between30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and80%, between 30% and 90%, or more, of the sum of the activities of saidcompstatin analog moieties.

The term “cell penetrating moiety” (CPM) refers to an entity that can beinternalized by a living cell and is capable of delivering or enhancingdelivery of a cargo to the interior of the cell. A cargo may be, e.g., apeptide, protein, nucleic acid, small molecule, or nanoparticle or otherentity of similar dimensions. The term “internalized by a cell” refersto gaining access to the interior (inside) of the cell. The “interior ofa cell” refers to locations within the boundary of the plasma membrane.For purposes hereof, membrane-bound intracellular vesicles and theircontents are considered to be inside the cell. Internalization may occurvia endocytotic processes and/or non-endocytotic processes (e.g.,pinocytosis, direct penetration, and transporter-mediated uptake) invarious embodiments. Entities that are contained in vesicles inside thecell, e.g., following endocytosis or pinocytosis, may be released fromsuch vesicles and enter the cytoplasm by various routes. For example,such entities may undergo retrograde transport from vesicles to theinterior of the endoplasmic reticulum (ER) and translocate from thereinto the cytoplasm or may directly translocate across vesicularmembranes.

In some embodiments a CPM is capable of entering cells of at least 5different cell types. In some embodiments a CPM is capable of enteringepithelial cells. It will be appreciated that “epithelium” refers tolayers of cells that line the cavities and surfaces of numerousstructures in the body and is the type of tissue from which many glandsare at least partly formed. Epithelial tissues include, for example,tissues found in the respiratory tract (e.g., nasal passages, trachea,bronchioles, bronchi, lungs), skin, gastrointestinal tract (e.g.,stomach, small intestine, colon), liver, biliary tract, pancreas,kidneys, ovaries, breast, prostate, cervix, uterus, bladder, ureter,testes, exocrine glands, endocrine glands, blood vessels, lymph vessels,etc. In some embodiments a CPM is capable of entering respiratoryepithelial cells. In some embodiments a CPM is capable of enteringretinal pigment epithelial (RPE) cells. In some embodiments a CPM iscapable of entering endothelial cells. In some embodiments a CPM iscapable of entering immune system cells, e.g., lymphocytes (B cells, Tcells, NK cells), granulocytes (e.g., neutrophils, eosinophils,basophils), mast cells, monocytes, and/or macrophages. In someembodiments a CPM is capable of entering nervous system cells, e.g.,neural cells, glial cells.

In some embodiments a cell penetrating moiety comprises a cellpenetrating peptide (CPP), sometimes referred to as a “proteintransduction domain”. Such peptides can be internalized by a cell anddelivering or enhancing delivery of a cargo to the interior of the cell.Naturally occurring CPPs occur in a number of different naturallyoccurring proteins including various viral proteins, animal proteins(e.g., insect, mammalian), and plant proteins. CPPs have been identifiedin certain secreted proteins, transcription factors, venoms, and toxins,among others. They are typically linear sequences ranging from about 6to about 30 amino acids in length that are able to mediate transport ofthe protein in which they occur into cells. In some embodiments a CPPcomprises or consists of such a naturally occurring amino acid sequence.In some embodiments a CPP comprises or consists of a non-naturallyoccurring amino acid sequence, i.e., an amino acid sequence not known tooccur in nature either alone or as part of a longer polypeptide. Anon-naturally occurring CPP may comprise a variant of a naturallyoccurring CPP, a chimeric sequence comprising portions of two or morenaturally occurring CPPs, or a sequence designed to have one or moreproperties of a naturally occurring CPP wherein such property correlateswith and/or is believed to be at least in part responsible for theability of the naturally occurring CPP to be internalized by a celland/or to enter a particular subcellular compartment (e.g., thecytoplasm) or organelle. In some embodiments a CPP is derived from adifferent CPP or from a polypeptide (e.g., a naturally occurringpolypeptide able to enter cells). For purposes of this disclosure, a CPPis considered to be “derived from” a particular polypeptide if the CPP(i) comprises or consists of a fragment of the polypeptide, wherein thefragment is at least 6 amino acids long, e.g., 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 amino acids long; (ii) comprises or consists of a peptide that is atleast 70% identical to a fragment of the polypeptide that is at least 10amino acids long; (iii) comprises or consists of a peptide whosesequence can be generated by making no more than 3 alterations (whichmay be substitution(s), deletion(s), or addition(s), in any combination)to the sequence of a fragment of the protein that is at least 10 aminoacids long; (iv) comprises or consists of a peptide that is a cyclized,circularly permuted, inverso, retro, or retro-inverso version of apeptide as described in any of (i), (ii), or (iii). In certainembodiments any of the peptides of (i), (ii), (iii), or (iv) may haveone or more modifications to one or more side chains, backbone, and/orto an N- or C-terminus. As will be appreciated, an inverso version of apeptide is the D-enantiomer of the peptide and has the same sequence asthe peptide but is composed of D-amino acids and has a mirrorconformation; a retro version of a peptide consists of the same sequenceof L amino acids but in reverse order; a retro-inverso version of apeptide consists of D-amino acids in the reverse order and is theD-retro-enantiomer of the peptide. In some embodiments a cellpenetrating moiety may be related to a CPP in that the CPM is designed,generated, derived, etc., from or based on the CPP, e.g., using a designprinciple or experimental approach intended to preserve, mimic, enhance,or select for ability to be internalized by a cell and/or to enter aparticular subcellular compartment (e.g., the cytoplasm or anorganelle).

In general, many CPPs may be broadly classified as cationic,hydrophobic, or amphipathic peptides. The term “cationic peptide” refersto a peptide that has a positive average net charge when in water at aphysiological pH, e.g., a pH of 7.0-7.4. In some embodiments a CPPcomprises or consists of a cationic peptide at least 6 amino acids long,e.g., 6-12, 12-20, or 20-30 amino acids long. In some embodiments atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the amino acids in acationic peptide are basic amino acids. In some embodiments a basicamino acid comprises a side chain that has a pK_(a) of at least 8.0, atleast 9.0, or at least 10.0 in water. In general, a basic amino acid maybe a standard amino acid or a non-standard amino acid. In someembodiments a basic amino acid comprises a side chain comprising aprimary or secondary amine or a guanidinium group. In some embodimentsat least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the amino acidsin a cationic peptide are independently selected from arginine,ornithine, lysine, and basic analogs of any of these. In someembodiments at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of theamino acids in a cationic peptide are independently selected fromarginine, lysine, and basic analogs of either of these. In someembodiments at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of theamino acids in a cationic peptide are arginine or lysine. In someembodiments at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of theamino acids in a cationic peptide are arginine. A basic analog of abasic amino acid may comprise substituent(s) at any one or morepositions, so long as the resulting compound retains a net positivecharge. In some embodiments a substituent is a lower alkyl or loweralkanoyl group.

In some embodiments a CPP comprises a hydrophobic peptide at least 6amino acids long, e.g., 6-12, 12-20, or 20-30 amino acids long. Ingeneral, a hydrophobic peptide is composed predominantly of hydrophobicand neutral amino acids. In some embodiments a hydrophobic peptidecomprises at least 50%, 60%, 70%, 80%, 90%, or more hydrophobic andneutral amino acids. A neutral amino acid may be selected from alanine,isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine,cysteine, methionine, threonine, glycine, serine, glutamine, and neutralanalogs thereof. Unless otherwise indicated or evident from the contextor use, “neutral” refers to neutral (uncharged) within a physiologicalpH range, e.g., between 7.0 and 7.4. A neutral analog of an amino acidmay comprise a neutral substituent as compared with the amino acid ofwhich it is an analog. A hydrophobic amino acid may be selected fromalanine, isoleucine, leucine, valine, phenylalanine, tryptophan,tyrosine, cysteine, methionine, and hydrophobic analogs of any of theforegoing nine amino acids, wherein the hydrophobicity of a hydrophobicanalog falls within the range of hydrophobicities of the foregoing nineamino acids or exceeds the upper limit of the range when measured usingthe same or substantially the same method and conditions as used todetermine the range. In some embodiments a hydrophobic analog is anamino acid that has increased hydrophobic character as compared with theamino acid of which it is an analog. Increased hydrophobic charactermay, for example, result from presence of one or more additionalhydrophobic groups or atoms in a side chain. In general, a hydrophobicgroup may be unsubstituted or substituted, provided that thesubstituent(s), if present, are sufficiently hydrophobic so as to notreduce the overall hydrophobicity of the amino acid below the lowerlimit of the afore-mentioned range. In some embodiments a hydrophobicgroup comprises or consists of an alkyl group, alkoxy group, ormonocyclic or bicyclic aromatic ring. In some embodiments an alkyl groupis a lower alkyl, e.g., methyl or ethyl. In some embodiments an alkoxygroup is a lower alkoxy, e.g., methoxy or ethoxy. In some embodimentsincreased hydrophobic character results from presence of one or moreadditional —CH₂— groups in an alkyl chain. In some embodiments ahydrophobic group or substituent comprises a halogen. In someembodiments a hydrophobic substituent is present at one or more atomsthat form part of the peptide backbone. In some embodiments ahydrophobic peptide comprises at least a portion of a signal sequence. Anumber of examples of hydrophobic peptides are provided herein. Peptideor amino acid hydrophobicity may be measured using a variety of methods.In some embodiments reverse phase HPLC may be used. For example, RP-HPLCmay be performed using a C8 column with 10 mM aqueous NaH₂P04, adjustedto pH 7 with NaOH and containing 50 mM NaCl as eluent A and using 50%acetonitrile containing 50 mM NaCl as eluent B. When comparinghydrophobicities of two or more peptides or amino acids, thehydrophobicities may be measured using the same method and same orapproximately the same conditions (e.g., pH, salt concentration, buffer)may be used, or different methods that are expected to giveapproximately the same results may be used. In some embodiments ahydrophobic peptide comprises at least a portion of a signal sequence(discussed further below).

The term “amphipathic peptide” refers to a peptide that possesses atleast one hydrophilic region and at least one hydrophobic region. Insome embodiments the hydrophilic and hydrophobic regions are present indistinct portions of the peptide sequence (primary structure). In someembodiments a CPP comprises an amphipathic peptide at least 6 aminoacids long e.g., 6-9, 9-12, 12-20, or 20-30 amino acids long. In someembodiments the sequence of an amphipathic peptide comprises at leastone sequence at least 4 amino acids long composed predominantly ofhydrophilic amino acids and at least one sequence at least one sequenceat least 4 amino acids long composed predominantly of hydrophobic aminoacids. In some embodiments the amphiphilic character of an amphipathicpeptide results at least in part from its secondary structure. Forexample, in some embodiments an amphipathic peptide comprises a helix,e.g., an alpha helix, having predominantly hydrophilic amino acidresidues aligned along one side of the helix and predominantlyhydrophobic amino acid residues aligned along the opposite side. Theterm “predominantly” is used to mean at least 75%, 80%, 85%, 90%, 95%,or 100%. Presence of a helix, e.g., an alpha helix, may be determinedexperimentally (e.g., spectrophotometrically, e.g., by circulardichroism spectroscopy in the far-ultraviolet (far-UV) spectral region(170-250 nm) or infrared spectroscopy), or may alternately oradditionally be predicted using various computer programs or algorithms,such as the Chou-Fasman algorithm (Chou, P. Y., et al. (1974)Biochemistry, 13: 222-45) or a modified version thereof (see, e.g., ChenH, Gu F, Huang Z (2006). “Improved Chou-Fasman method for proteinsecondary structure prediction”. BMC Bioinformatics 7 (Suppl 4): SI4),or other suitable algorithms or programs known in the art.

In some embodiments a CPP is both cationic and amphipathic.

In some embodiments a CPP comprises or is derived from at least aportion of a signal sequence. Signal sequences, also referred to assignal peptides, are typically between 5 to about 30 amino acids longand are found in most polypeptides that are destined for the secretorypathway. Such proteins include proteins that are secreted from the cellor reside within certain organelles (the endoplasmic reticulum, golgi,or endosomes) or are inserted into various cellular membranes (e.g., theplasma membrane). Examples of secreted proteins include, e.g., variousgrowth factors, cytokines, clotting factors, and other proteins found inthe blood or extracellular space. Examples of membrane proteins include,e.g., receptors, channels, transporters, and various proteins thatinteract with such proteins. In secreted proteins and a number oftransmembrane proteins, a signal sequence is typically located at theN-terminus and is cleaved from the nascent polypeptide once it has beentranslocated into the membrane of the ER (in eukaryotes) or plasmamembrane (in prokaryotes) by signal peptidase. A polypeptide thatcomprise a signal sequence that (at least under typical conditions) issubsequently removed and is therefore absent in the mature, functionalform of the polypeptide may be referred to as a “precursor polypeptide”.Typical signal peptides contain a hydrophobic domain, which may becomposed predominantly of hydrophobic amino acids and may form analpha-helix. In some embodiments a CPP comprises or is derived from atleast a portion of such a hydrophobic domain of a signal sequence. Manysignal peptides begin with a short positively charged stretch of aminoacids. A stretch of amino acids that is recognized and cleaved by signalpeptidase is typically located at the end of the signal peptide. Signalsequences of certain membrane and transmembrane proteins are not cleavedoff but rather serve to anchor the protein to the membrane and thereforeare sometimes referred to as signal anchor sequences. They may belocated, e.g., in the first transmembrane domain. Signal sequences ofnumerous secreted and membrane-bound polypeptides have been determinedexperimentally and/or predicted based on the sequence. Signal peptidesmay be annotated in database records (e.g., GenBank records) for theprotein. In some embodiments a CPP is derived from the hydrophobicdomain of the human fibroblast growth factor 4 (FGF4) signal sequence.

A CPP may be linear or cyclic. A cyclic CPP may be cyclized via a bondbetween the N- and C-termini, a bond between a terminus and a sidechain, a bond between two side chains, or a bond between the backboneand a side chain. In some embodiments a CPP may be cyclized via a linkerthat forms a bond to each terminus, to a terminus and a side chain, totwo side chains, or to the backbone and a side chain. In someembodiments the linker comprises or consists of a peptide.

Table 2 lists a variety of CPPs that may be used in various embodiments.The numbers in parentheses following a protein name herein indicate thefirst and last amino acids in a fragment of the protein. For example,Tat (49-56) refers to a peptide whose sequence consists of amino acids49-56 of Tat. In some embodiments a CPP that is derived from or relatedto a CPP listed in Table 2 may be used. Table 2 also provides in someinstances, whether a peptide is composed of L or D amino acids, the nameof a peptide or protein in which a CPP is found or from which a CPP isderived. Lower case letters represents D-amino acids.

TABLE 2 Various CPPs GRKKRRQRRRPPQ L Tat (48-60) HIV-1 GISYGRKKRRQRRRPPQL Tat (43-60) HIV-1 FITKALGISYGRKKRRQRRRPPQ L Tat (37-60) HIV-1GRKKRRQRRR L Tat (48-57) HIV-1 RKKRRQRRR L Tat (49-57) HIV-1 RKKRRQRRL Tat (49-56) HIV-1 rkkrrqrrr D D-Tat (49-57) HIV-1 RRRQRRKKRL Retro - Tat (57-49) HIV-1 rrrqrrkkr D D-Tat (57-49) HIV-1 RKKRRARRRL Ala54 substitution mutant of Tat (49-57) HIV- 1 GRKKRRQRRRCL Pro deletion mutant of Tat (48-60) HIV-1 TRQARRNRRRRWRERQRL Rev (34-50) HIV-1 GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS L GalaninINLKALAALAKKIL L MP Wasp venom peptide Mastoparan RQIKIWFQNRRMKWKKL Antennapedia homeodomain of drosophila RQIKIWFQNRRMKWKKL pAntpHD (43-58) Antennapedia KKWKMRRNQFWIKIQRL pAntpHD (5 8-43) Antennapedia rqikiwfqnrrrnkwkk DD form of pAntpHD (43-58) Antennapedia RQIKTWFPNRRMKWKKL pAntpHD (Pro50) Antennapedia RQPKIWFPNRRKPWKKL pAntpHD (3Pro) Antennapedia RQIKIWFQNRRMKWKKL pAntp (43-58) Antennapedia RQIKIWFQNRRMKWKL pAntp (43-57) Antennapedia RQIKIWFQNRRMKW L pAntp (43-56) AntennapediaIKIWFQNRRMKWKK L pAntp (45-58) Antennapedia RQIKIWFPNRRMKWKKL Penetratin (pAntp)(43-58) Antennapedia RAAARQARAG L PTD4 YARAAARQARAGL PTD4 KMDCRWRWKCCKK L Crot (27-39) Retal snake venom (Crotamine)RKKRRRESRKKRRRES L DPV3 Human Superoxide dismutase GRPRESGKKRKRKRLKPL DPV6 Human platelet-derived growth factor GKRKKKGKLGKKRDPL DPV7 Human Epidermal-like growth factor SRRARRSPRESGKKRKRKR L DPV10/6VPMLK L Bip1 Bax-binding domain of human Ku70 KLPVML Bip9 Bax-binding domain of human Ku70 TKRRITPKDVIDVRSVTTEINTL Inv3 Mycobacterium cell entry protein (McelA)AEKVDPVKLNLTLSAAAEALTGLGDK L Inv5 Mycobacterium cell entry protein(McelA TKRRITPKDVIDVRSVTTKINT L Inv3.5 Mycobacterium cell entry protein(McelA) KLIKGRTPIKFGKADCDRPPKHSQNGMGK L Res 1 L3 loop of restrictocinKRIPNKKPGKKTTTKPTKKPTIKTTKKDLKPQTTKPK L RSV-A1 Human respiratorysyncytial virus, type A DRRRRGSRPSGAERRRRRAAAAL RSG 1.2 Arg-rich peptide GTKMIFVGIKKKEERADLIAYLKKAL Cyt C 71-101 Human Cytochrome C RRRRNRTRRNRRRVRGCL FHV coat (35-49) RNA Binding Peptides MITYRDLISKKL TCTP-CPP 1 Human translationally controlled tumor protein MIIYRDKKSHL TCTP-CPP 2 Human translationally controlled tumor protein MIIFRDLISHL TCTP-CPP 3 Human translationally controlled tumor protein MITYRDLISHL TCTP Human translationally controlled tumor protein RRRRRRRR L R8RRRRRRRRR L R9 rrrrr D D-R6 rrrrrrr D D-R7 rrrrrrrr D D-R8rrrrrrrrr D D-R9 GWTLNSAGYLLGKINLKALAALAKKIL L Transportan (TP)ALWKTLLKKVLKAPKKKRKV L S4( 13)-PV Dermaseptin S4 peptide + SV40 NLSEEEAAGRKRKKRT L Glu-October-6 Transcription factor October-6 basedchimeric peptide KETWWETWWTEWSQPKKKRKV L Pep-1 GLRRLRQRRRLRRERVRAL human neurturin AAVALLPAVLLALLAP L KWKLFKKIGAVLKVL L KKLFKKILKYL L

In some embodiments a CPP is derived from or related to the Tat proteinof human immunodeficiency virus (HIV), e.g., Tat protein of HIV-1. Forexample, in some embodiments a CPP comprises or consists of a fragmentof Tat comprising Tat (49-56). For example in some embodiments a CPPcomprises or consists of Tat (49-56), Tat (49-57), Tat (48-56), Tat(48-57), Tat (47-56), Tat (47-57), or a retro-inverso version of any ofthese.

In some embodiments a CPP is related to or derived from the Rev proteinof human immunodeficiency virus, e.g., Rev protein of HIV-1.

In some embodiments a CPP is derived from or related to a transcriptionfactor. In some embodiments the transcription factor is a homeodomainprotein. In some embodiments a CPP comprises or is derived from orrelated to at least a portion of a homeodomain, e.g., at least a portionof the third helix of a homeodomain. In some embodiments the homeodomainprotein is Antennapedia protein, e.g., of Drosophila, e.g., Drosophilamelanogaster. In some embodiments the homeodomain protein is the Isl-1protein. In some embodiments the homeodomain protein is knotted-1, e.g.,of maize.

In some embodiments a CPP is derived from or related to crotamine(Crot). Crotamine is a 42-residue polypeptide derived from the venom ofthe South American rattlesnake Crotalus durissus terrificus. Forexample, in some embodiments a CPP comprises or consists of a fragmentof crotamine. In some embodiments a CPP comprises or consists of Crot(30-39), also known as CyLoPl, or is derived from or related to CyLoPl.

In some embodiments a CPP comprises or is derived from or related to anantimicrobial peptide (AMP). Many AMPs possess two functionallyimportant properties, namely a net positive charge (cationic) and theability to assume an amphipathic structure. Numerous AMPs have beenisolated (see, e.g., the list available athttp://w\vw.bbcm.univ.trieste.iL'˜tossi/pagl.htm) and many additionalAMPs have been designed and synthesized based on naturally occurringAMPs. For example, cecropins, first isolated from the hemolymph of thegiant silk moth Hyalophora cecropia, are a family of peptides composedof 31-39 amino acids with antibacterial activity against bothGram-negative and Gram-positive bacteria. Melittin, another AMD, is theprincipal active component of apitoxin (bee venom). Additional examplesof AMPs include, e.g., magainin and buforin 2. In some embodiments a CPPcomprises BPOO or is derived from or related to Pep3 or BPOO. Pep3(CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2) and BPlOO (KKLFKKILKYL-NH2) arececropin A-melittin hybrid peptides.

In some embodiments a CPP is derived from or related to a sequence foundin a human protein. In some embodiments the human protein istranslationally-controlled tumor protein 1, neurturin, FGF4, calcitonin,period 1 circadian protein, superoxide dismutase, or secretoryleukoprotease inhibitor. It will be appreciated that in some embodimentsthe sequence may also be found in one or more non-human species, e.g.,one or more non-human mammalian species.

In some aspects, the present invention provides a polypeptide comprisinga compstatin analog sequence and a sequence listed in Table 2. In someaspects, the present invention provides a polypeptide comprising acompstatin analog sequence and a sequence derived from a sequence listedin Table 2. In some embodiments the polypeptide may comprise any of SEQID NOs: 3, 4, 5, or 6, and any of the sequences listed in Table 2 or anysequence derived therefrom. In some embodiments the polypeptide maycomprise any of SEQ ID NOs: 9-36 and any of the sequences listed inTable 2 or any sequence derived therefrom. In some embodiments thecompstatin analog sequence is located N-terminal to the sequence from orderived from Table 2. In some embodiments the compstatin analog sequenceis located C-terminal to the sequence in or derived from a sequence inTable 2. In some embodiments one or more amino acids is positionedbetween the compstatin analog sequence is located C-terminal to thesequence in or derived from a sequence in Table 2. In some embodimentsAEEAc is positioned between the compstatin analog sequence is locatedC-terminal to the sequence in or derived from a sequence in Table 2. Anysuch polypeptide may comprise a blocking moiety at the N-terminus,C-terminus, or both.

In some embodiments L comprises one or more amino acids. For example, Lmay be a single amino acid or a peptide comprising, e.g., between 2 and5, 5 and 10, or 10 and 20 amino acids. In some embodiments a CPCA isbetween 18 and 25 amino acids, between 25 and 35 amino acids, or between25 and 50 amino acids in length. In certain embodiments a CPCA is 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. Incertain embodiments wherein A is a peptide (e.g., a CPP), L is absent orcomprises an amino acid or peptide, a CPCA may be produced using solidphase peptide synthesis procedures known in the art. In someembodiments, for example, L comprises or consists of AEEAc.

In some embodiments a CPP is joined directly to the N- or C-terminalamino acid of a compstatin analog by a peptide bond. The structure ofthe resulting compound may be represented as CPP-CA or CA-CPP, where“CA” represents a compstatin analog. In some embodiments a CPP is joinedto a compstatin analog via a linker. In some embodiments the linkercomprises one or more amino acids. In some embodiments the linker is apeptide. The linker may be joined to the N- or C-terminus of thecompstatin analog or to a side chain in various embodiments. In someembodiments the linker is an amino acid or peptide, the N-terminus ofthe linker is joined to the C-terminus of either the CPP or the CA, andthe C-terminus of the linker is joined to N-terminus of the CA or CPP,respectively. The structure of the resulting CPCA may be represented asCPP-(Xaa)n-CA or CA-(Xaa)n-CPP, where (Xaa)n is an amino acid or aminoacid sequence and wherein each Xaa may be any amino acid. In someembodiments the number of amino acids (n) in the linker is between 1 and5, between 1 and 10, between 5 and 10, between 10 and 15, 15 and 20, or20 and 25. Typically n is relatively small, e.g., between 1 and 15,though longer sequences, e.g., between 25 and 50, 50 and 100, or 100 and1,000 may be used in certain embodiments. In some embodiments (Xaa)n iscomposed of standard amino acids. In some embodiments (Xaa)n comprisesat least one non-standard amino acid. In some embodiments (Xaa)ncomprises at least one standard amino acid and at least one non-standardamino acid. In some embodiments (Xaa)n is composed of non-standard aminoacids. In some embodiments n is 1 and Xaa is glycine. In someembodiments n is 1 and Xaa is AEEAc (amino-3, 6-dioxaoctanoic acid, alsoknown as 2-[−2-(−amino)-ethoxy]-ethoxy}-acetic acid). The CPCA may insuch cases be represented as CPP-AEEAc-CA or CA-AEEAc-CPP. In someembodiments n is greater than 1, e.g., between 1 and 5, and (Xaa)ncomprises at least one AEEAc. In some embodiments (Xaa)n comprises atleast one neutral or hydrophobic amino acid. In some embodiments (Xaa)ncomprises at least one neutral or hydrophobic amino acid and AEEAc. Forexample, in some embodiments (Xaa)n comprises or consists of L-AEEAc,LC-AEEAc, CL-AEEAc, I-AEEAc, IC-AEEAc, CI-AEEAc, or LIC-AEEAc. In someembodiments each Xaa is independently glycine, serine, or AEEAc. In someembodiments each Xaa is independently neutral or hydrophobic amino acidor AEEAc. In some embodiments in which a CPP is joined directly to theN- or C-terminal amino acid of a compstatin analog by a peptide bond orin which a CPP is joined to a compstatin analog by peptide linker, theCPCA is produced using standard solid phase peptide synthesistechniques.

In some embodiments a CPP-CA or CA-CPP sequence lacks predicted MHCClass I epitopes and/or lacks predicted or experimentally confirmed MHCClass II epitopes. In some embodiments a CPP-(Xaa)n and/or (Xaa)n-CPPsequence lacks predicted or experimentally confirmed MHC Class Iepitopes and/or lacks predicted MHC Class II epitopes. In someembodiments a CPP-(Xaa)n-CA and/or CA-(Xaa)n-CPP sequence lackspredicted MHC Class I epitopes and/or lacks predicted or experimentallyconfirmed MHC Class II epitopes. In some embodiments a CPP-(Xaa)n-CAand/or CA-(Xaa)n-CPP sequence lacks predicted MHC Class II epitopes aspredicted by TEPITOPE or TEPITOPEPan, available athttp://www.biokdd.fudan.edu.cn/Service/TEPITOPEpan/ ((Zhang, et al,TEPITOPEpan: Extending TEPITOPE for Peptide Binding Prediction Coveringover 700 HLA-DR Molecules, PLoS ONE 7(2): e30483.doi:10.1371/journal.pone.0030483).

In some embodiments a CPM-CA, when injected into a mammal, e.g., arabbit, non-human primate, or human according to a standard protocolsuitable for generating antibodies to a typical protein, does not resultin production of antisera that are capable of detecting the CPM-CA byWestern blot above the level of control antisera from an unimmunizedanimal or an animal immunized with a suitable control peptide, e.g., apeptide of unrelated sequence or a scrambled sequence of the same aminoacids that does not share any subsequences greater than 3 amino acidslong. Examples of standard immunization protocols are found, e.g., inAusubel, Current Protocols in Immunology, supra and/pr Harlow, supra.

In some embodiments a cell penetrating moiety may comprise or consist ofa poly-N-substituted amino acid, e.g., a poly-N-alkylated amino acid. Insome embodiments a poly-N-substituted amino acid comprisespoly-N-substituted glycine, e.g., poly-N-alkylated amino acid sometimestermed an -peptoid. As known in the art, such compounds are composed ofresidues in which the side chain is attached to the nitrogen atom of thepeptide backbone, rather than to the a-carbon as it is in the standardamino acids. Poly-N-substituted amino acids include, e.g., N-alkylatedβ-alanine (sometimes termed-peptoids); α,β-alternating peptoids;α,β-mixed peptoids, extended peptoids, or oligomers comprising othervariant backbone amino-acid monomer units. Side chains present in apoly-N-substituted amino acid may, for example, be among those found instandard and/or non-standard-amino acids. In some embodiments a cellpenetrating moiety comprises two or more peptide architectures, e.g.,containing a backbone comprising or consisting both of alpha- andbeta-amino acids (alpha/beta-peptides) or comprising or consisting bothof alpha-amino acids and N-alkylated amino acids. In some embodiments acell penetrating moiety comprises alpha-amino acids and N-alkylatedβ-alanine residues. Different types of amino acids may be present in anyproportion or arrangement. For example, a peptide may comprise two ormore segments having different peptide architectures or may comprisealternating types of residues. For example, in some embodiments a cellpenetrating moiety comprises alternating N-alkylated β-alanine anda-amino acid residues. Further information regarding peptoids, peptoidvariants, and/or hybrid molecules is found e.g., in Simon, R J, et al.“Peptoids: a modular approach to drug discovery” Proceedings of theNational Academy of Sciences USA, (1992), 89(20), 9367-9371; U.S. Pat.No. 5,811,387; Yoo, B. and Kirshenbaum, K., Peptoid architectures:elaboration, actuation, and application. Curr. Opin. Chem. Biol. (2008),12, 714-721; Culf, A S and Ouellette, R J, Solid-Phase Synthesis ofN-Substituted Glycine Oligomers (a-Peptoids) and Derivatives, Molecules(2010), 15, 5282-5335; Olsen C A, Peptoid-Peptide hybrid backbonearchitectures. Chembiochem. (2010), 11(2): 152-60. The references citedin these references are incorporated herein by reference.

In general, linking portion L can comprise any one or more aliphaticand/or aromatic moieties consistent with the formation of a stablecompound joining the linked moieties. The term “stable”, as used herein,preferably refers to compounds which possess stability sufficient toallow manufacture and which maintain the integrity of the compound for asufficient period of time, e.g., to be useful for one or more purposesdescribed herein. In some embodiments, L comprises a saturated orunsaturated, substituted or unsubstituted, branched or unbranched,aliphatic chain having a length of between 1 and 50, between 1 and 40,between 1 and 30, between 1 and 20, between 1 and 10, between 1 and 6,or 5 or less carbon atoms, where length refers to the number of C atomsin the main (longest) chain. In some embodiments, the aliphatic chaincomprises one or more heteroatoms (O, N, S), which may be independentlyselected. In some embodiments, at least 50% of the atoms in the mainchain of L are carbon atoms. In some embodiments, L comprises asaturated alkyl moiety (CH₂)_(n), wherein n is between 1 and 30.

In some embodiments, L comprises one or more heteroatoms and has alength of between 1 and 50, between 1 and 40, between 1 and 30, between1 and 20, or between 1 and 10 total carbon atoms in a chain, or less. Insome embodiments, L comprises an oligo(ethylene glycol) moiety(-(0-CH₂—CH₂-)_(n)) wherein n is between 1 and 50, between 1 and 40,between 1 and 30, between 1 and 20, or between 1 and 10. In someembodiments, L comprises an unsaturated moiety such as —CH═CH— or—CH₂—CH═CH—; a moiety comprising a non-aromatic cyclic ring system(e.g., a cyclohexyl moiety), an aromatic moiety (e.g., an aromaticcyclic ring system such as a phenyl moiety); an ether moiety (—C-0-C—);an amide moiety (—C(=0)-N—); an ester moiety (—CO-0-); a carbonyl moiety(—C(=0)-); an imine moiety (—C═N—); a thioether moiety (—C—S—C—); anamino acid residue; and/or any moiety that can be formed by the reactionof two compatible reactive functional groups. In certain embodiments,one or more moieties of a linking portion is/are substituted byindependent replacement of one or more of the hydrogen (or other) atomsthereon with one or more moieties including, but not limited toaliphatic; aromatic, aryl; alkyl, aralkyl, alkanoyl, aroyl, alkoxy;thio; F; CI; Br; I; —N02; —CN; —CF3; —CH2CF3; —CHC12; —CH20H; —CH2CH20H;—CH2NH2; —CH2S02CH3; — or -GRGI wherein G is -0-, —S—, —NRG2-, —C(=0)-,—S(=0)-, —S02-, —C(=0)0-, —C(=0)NRG2-, —OC(=0)-, —NRG2C(=0)-, —OC(=0)0-,—OC(=0)NRG2-, —NRG2C(=0)0-, —NRG2C(=0)NRG2-, —C(═S)—, —C(═S)S—,—SC(═S)—, —SC(═S)S—, —C(=NRG2)-, —C(═NRG2)0-, —C(═NRG2)NRG3-,—OC(═NRG2)-, —NRG2C(=NRG3)-, —NRG2S02-, —NRG2S02NRG3-, or —S02NRG2-,wherein each occurrence of RG1, RG2 and RG3 independently includes, butis not limited to, hydrogen, halogen, or an optionally substitutedaliphatic, aromatic, or aryl moiety. It will be appreciated that cyclicring systems when present as substituents may optionally be attached viaa linear moiety. Combinations of substituents and variables envisionedby this invention are preferably those that result in the formation ofstable compounds useful in any one or more of the methods describedherein, e.g., useful for the treatment of one or more disorders and/orfor contacting a cell, tissue, or organ, as described herein, and/oruseful as intermediates in the manufacture of one or more suchcompounds.

In some embodiments multiple linking portions can be attached to oneanother to form a larger linking portion L, and at least some of suchlinking portions can have one or more compstatin analog moietie(s)attached thereto. In molecules comprising multiple compstatin analogmoieties, the compstatin analogs can be the same or different and, ifdifferent, can be independently selected. The same applies to thelinking portions and cell-penetrating groups. In various embodiments theinvention encompasses the synthesis and use of multivalent compstatinanalogs comprising one or more cell-penetrating group(s) and use ofconcatamers of compstatin analogs comprising one or morecell-penetrating group(s). In some embodiments, a linkage is a stablenon-covalent linkage such as a biotin/(strept)avidin linkage or othernoncovalent linkage of approximately equivalent strength. In someembodiments a linkage comprises a moiety that is susceptible tointracellular cleavage in at least some cells or under at least someintracellular conditions. In some embodiments a cleavage-susceptiblelinkage comprises a cleavage site for a protease that is activeintracellularly under at least some conditions. Without wishing to bebound by any theory, intracellular cleavage may in some embodimentsenhance the ability of a compstatin analog to bind to C3 or C3bintracellularly.

In some embodiments a cell-penetrating compstatin analog comprises acompstatin analog in which any of SEQ ID NOs: 3-36, 37A, 38A, 39A, 40A,or 41A is extended by one or more amino acids at the N-terminus,C-terminus, or both, wherein in some embodiments at least one of theamino acids has a side chain that comprises a reactive functional groupsuch as a primary or secondary amine, a sulfhydryl group, a carboxylgroup (which may be present as a carboxylate group), a guanidino group,a phenol group, an indole ring, a thioether, or an imidazole ring. Insome embodiments, the amino acid(s) is/are L-amino acids. In someembodiments, any one or more of the amino acid(s) is a D-amino acid. Ifmultiple amino acids are added, the amino acids can be independentlyselected. In some embodiments, the reactive functional group (e.g., aprimary or secondary amine) is used as a target for addition of a moietycomprising a cell penetrating moiety. Amino acids having a side chainthat comprises a primary or secondary amine include lysine (Lys) anddiaminocarboxylic acids of general structure NH₂(CH₂)_(n)CH(NH₂)COOHsuch as 2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid(daba), and ornithine (orn), wherein n=1 (dapa), 2 (daba), and 3 (orn),respectively. In some embodiments at least one amino acid is cysteine,aspartic acid, glutamic acid, arginine, tyrosine, tryptophan,methionine, or histidine. Cysteine has a side chain comprising asulfhydryl group. Aspartic acid and glutamic acid have a side chaincomprising a carboxyl group (ionizable to a carboxylate group). Argininehas a side chain comprising a guanidino group. Tyrosine has a side chaincomprising a phenol group (ionizable to a phenolate group). Tryptophanhas a side chain comprising an indole ring include, e.g., tryptophan.Methionine has a side chain comprising a thioether group include, e.g.,methionine. Histidine has a side chain comprising an imidazole ring. Awide variety of non-standard amino acids having side chains thatcomprise one or more such reactive functional group(s) are available,including naturally occurring amino acids and amino acids not found innature. See, e.g., Hughes, B. (ed.), Amino Acids, Peptides and Proteinsin Organic Chemistry, Volumes 1-4, Wiley—VCH (2009-2011); Blaskovich,M., Handbook on Syntheses of Amino Acids General Routes to Amino Acids,Oxford University Press, 2010. The invention encompasses embodiments inwhich one or more non-standard amino acid(s) is/are used to provide atarget for addition of a moiety of interest, such as a moiety comprisinga cell-penetrating group or a clearance-reducing moiety (discussedfurther below). Any one or more of the amino acid(s) may be protected asappropriate during synthesis of the compound. For example, one or moreamino acid(s) may be protected during reaction(s) involving the targetamino acid side chain. In some embodiments, wherein asulfhydryl-containing amino acid is used as a target for addition of amoiety of interest, such as a moiety comprising a cell-penetrating groupor clearance reducing moiety, the sulfhydryl is protected while thecompound is being cyclized by formation of an intramolecular disulfidebond between other amino acids such as cysteines.

In the discussion in this paragraph, an amino acid having a side chaincontaining an amine group is used as an example. The inventionencompasses analogous embodiments in which an amino acid having a sidechain containing a different reactive functional group is used. In someembodiments, an amino acid having a side chain comprising a primary orsecondary amine is attached directly to the N-terminus or C-terminus ofany of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A or via a peptidebond. In some embodiments, an amino acid having a side chain comprisinga primary or secondary amine is attached to the N- or C-terminus of anyof SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A or via a linkingportion, which may contain any one or more of the linking moietiesdescribed above. In some embodiments, at least two amino acids areappended to either or both termini. The two or more appended amino acidsmay be joined to each other by peptide bonds or at least some of theappended amino acids may be joined to each other by a linking portion,which may contain any one or more of the linking moieties describedherein. Thus in some embodiments, a cell-penetrating compstatin analogcomprises a compstatin analog moiety M of formula B1-R1-M i-R2-B2,wherein M i represents any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A,or 41A, either R1 or R2 may be absent, at least one of R1 and R2comprises an amino acid having a side chain that contains a primary orsecondary amine, and B1 and B2 are optionally present blocking moieties.R1 and/or R2 may be joined to M i by a peptide bond or a non-peptidebond. R1 and/or R2 may comprise a linking portion L^(P3). For example,R1 can have formula M2-L^(P3) and/or R2 can have formula L^(P3)-M2wherein L^(P3) is a linking portion, and M2 comprises at least one aminoacid having a side chain comprising a primary or secondary amine. Forexample, M2 can be Lys or an amino acid chain comprising Lys. In someembodiments, L^(P3) comprises of consists of one or more amino acids.For example, L^(P3) can be between 1 and about 20 amino acids in length,e.g., between 4 and 20 amino acids in length. In some embodiments,L^(P3) comprises or consist of multiple Gly, Ser, and/or Ala residues.In some embodiments, L^(P) ³ does not comprise an amino acid thatcomprises a reactive SH group, such as Cys. In some embodiments, L^(P) ³comprises an oligo(ethylene glycol) moiety and/or a saturated alkylchain. In some embodiments, L^(P) ³ is attached to the N-terminal aminoacid of Mi via an amide bond. In some embodiments, L^(P3) is attached tothe C-terminal amino acid of Mi via an amide bond. The compound may befurther extended at either or both termini by addition of furtherlinking portion(s) and/or amino acid(s). The amino acids can the same ordifferent and, if different, can be independently selected. In someembodiments, two or more amino acids having side chains comprisingreactive functional groups are used, wherein the reactive functionalgroups can be the same or different. The two or more reactive functionalgroups can be used as targets for addition of two or more moieties. Insome embodiments, two or more cell-internalizing moieties are added. Insome embodiments, a cell penetrating moiety and a clearance reducingmoiety are added. In some embodiments, a linker and/or cell penetratingmoiety is attached to an amino acid side chain after incorporation ofthe amino acid into a peptide chain. In some embodiments, a linkerand/or cell penetrating moiety is already attached to the amino acidside chain prior to use of the amino acid in the synthesis of acell-reactive compstatin analog. For example, a Lys derivative having alinker attached to its side chain can be used. The linker may comprise acell-penetrating functional group or may subsequently be modified tocomprise a cell-penetrating functional group.

Certain reactive compstatin analogs, which may be used to producecell-penetrating compstatin analogs, are described in further detailbelow. In some embodiments such reactive compstatin analogs may bereacted with a CPM comprising a compatible reactive functional group toproduce a cell-penetrating compstatin analog. In some embodiments suchreactive compstatin analogs may be reacted with a linker comprising acompatible reactive functional group, wherein the linker is or may beattached to a CPM, to produce a cell-penetrating compstatin analog. Inthe following discussion, a peptide having the amino acid sequenceIle-Cys*-Val-(lMe)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr (SEQ ID NO:37) (corresponding to the compstatin analog of SEQ ID NO: 28, whereinasterisks in SEQ ID NO: 37 represent cysteines joined by a disulfidebond in the active compound, and (lMe)Trp represents1-methyl-tryptophan)), is used as an exemplary compstatin analog moiety;maleimide (abbreviated Mai) is used as an example of a reactivefunctional group; (CH2)_(n) and (0-CH2-CH2)_(n) are used as examples oflinking portions; lysine is used as an example of an amino acidcomprising a reactive functional group (in some compounds), andacetylation and amidation of the N- and C-termini, respectively, areused as optionally present exemplary blocking moieties in some compoundsand are represented in italics, i.e., as Ac and NH₂ respectively. Itwill be appreciated that the compounds can be prepared using a varietyof synthetic approaches and using a variety of precursors. Thediscussion of various synthetic approaches and precursors below is notintended to limit the invention. In general, any of the features of anyof the compounds described below or herein can be freely combined withfeature(s) of other compounds described below or elsewhere herein, andthe invention encompasses such embodiments.

In some embodiments, a reactive moiety is provided by a reactivecompound comprising a maleimide group (as a reactive functional group)and an alkanoic acid (RCOOH), where R is an alkyl group. For example,6-malemeidocaproic acid (Mal-(CH2₎₅. COOH), depicted below, can be used.

In some embodiments, a reactive moiety is provided by a derivative of analkanoic acid in which the carboxylic acid moiety has been activated,e.g., the OH moiety has been converted to a better leaving group. Forexample, the carboxyl group of compound I may be reacted with EDC,followed by reaction with NHS (which can optionally be provided aswater-soluble sulfo-NHS), resulting in an N-hydroxysuccinimide esterderivative of 6-malemeidocaproic acid, i.e., 6-maleimidohexanoic acidN-hydroxysuccinimide (NHS) ester (depicted below).

The compound of SEQ ID NO: 37 can be modified at the N- and/orC-terminus to generate a reactive compstatin analog. For example,compound II can be used to generate the following reactive compstatinanalog by reaction with the N-terminal amino group of Ile.

Maleimide-(CH₂)₅—C(=0)-Ile-Cys*-Val-(Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH₂(SEQ ID NO: 38). It will be appreciated that in SEQ ID NO: 38 the —C(=0)moiety is attached to the immediately C-terminal amino acid (He), via aC—N bond, wherein the N is part of the amino acid and is not shown.

In other embodiments, a maleimide group is linked to Thr at theC-terminus, resulting in the following reactive compstatin analog:

(SEQ ID NO: 39) {circumflex over( )}c-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(C═0)-(CH₂)₅-maleimide.

In some embodiments, a reactive compstatin analog can be synthesizedusing bifunctional linker (e.g., a heterobifunctional linker). Anexemplary heterobifunctional linker comprising (CH₂—CH₂-0)_(n) and(CH₂)_(m) (where m=2) moieties is shown below:

Compound III comprises a maleimide group as a reactive functional groupand an NHS ester moiety that reacts readily with an amino group (e.g.,an N-terminal amino group or an amino group of an amino acid sidechain).

An embodiment of compound III in which n=2 can be used to generate thefollowing reactive compstatin analog using the compstatin analog of SEQID NO: 37:

(SEQ ID NO: 40) Maleimide-(CH₂)₂-C(═0)-NH-CH ₂CH₂OCH₂CH₂OCH₂CH₂C(═0)-Ile-Cys*-Val-(1Me)Trp -Gln-Asp-Trp-Gly-Ala- His-Arg-Cys*-Thr -NH₂

It will be appreciated that in the compound of SEQ ID NO: 40 a —C(=0)moiety is attached to the N-terminal amino acid (He residue via a C—Nbond, wherein the N is part of the amino acid and is not shown. In someembodiments a linker has the formula of Compound III wherein n≥1.Exemplary values for n in a (CH₂—CH₂-0)_(n) moiety are provided herein.

In some embodiments, the alkyl chain that links the maleimide moiety tothe rest of the molecule contains more or fewer methylene units, theoligo(ethylene glycol) moiety contains more or fewer ethylene glycolunits, and/or there are more or fewer methylene units flanking either orboth sides of the oligo(ethylene glycol) moiety, as compared with thecompound of SEQ ID NO: 39 or SEQ ID NO: 40. Exemplary reactivecompstatin analogs illustrative of a few such variations are presentedbelow (SEQ ID NOs: 41-46):

(SEQ ID NO: 41) Maleimide-(CH₂)₂-C(═0)-NH-CH ₂CH₂OCH₂CH₂C(═0)-Ile-Cys*-Val-(1Me)Trp -Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂(SEQ ID NO: 42) Maleimide-(CH₂)₃-C(═0)-NH-CH ₂CH₂OCH₂CH₂OCH₂C(═0)-Ile-Cys*-Val-(1Me)Trp -Gln-Asp-Trp-Gly-Ala-His- Arg-Cys*-Thr-NH₂(SEQ ID NO: 43) Maleimide-(CH₂)₅-C(═0)-NH-CH ₂CH₂OCH₂CH₂OCH₂C(═0)-Ile-Cys*-Val-(1Me)Trp -Gln-Asp-Trp-Gly-Ala-His- Arg-Cys*-Thr-NH₂(SEQ ID NO: 44) Maleimide-(CH₂)₄-C(═0)-NH-CH ₂CH₂OCH₂CH₂OCH₂CH₂C(═0)-Ile-Cys*-Val-(1Me)Trp -Gln-Asp-Trp-Gly-Ala- His-Arg-Cys*-Thr-NH₂(SEQ ID NO: 45) Maleimide-(CH₂)₂-C(═0)-NH-CH ₂CH₂OCH₂CH₂OCH₂CH₂C(═0)-Ile-Cys*-Val-(1Me)Trp -Gln-Asp-Trp-Gly-Ala- His-Arg-Cys*-Thr-NH₂(SEQ ID NO: 46) Maleimide-(CH₂)₅-C(═0)-NH-CH ₂CH₂OCH₂CH₂OCH₂C(═0)-Ile-Cys*-Val-(IMe)Trp -Gln-Asp-Trp-Gly-Ala-His- Arg-Cys*-Thr-NH₂

In some embodiments, SEQ ID NO: 37 is extended to comprise a Lys residueat the N- or C-terminus of the peptide, e.g., as exemplified below for aC-terminal linkage:

(SEQ ID NO: 47) {circumflex over( )}ic-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys -NH₂.

In some embodiments, a Lys residue is attached to the N- or C-terminusof SEQ ID NO: 37 via a peptide linker, e.g., as exemplified below for aC-terminal linkage:

(SEQ ID NO: 48) {circumflex over( )}c-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly) ₅-Lys-NH₂.

In some embodiments, a linker comprising a primary or secondary amine isadded to the N- or C-terminus of a compstatin analog. In someembodiments, the linker comprises an alkyl chain and/or anoligo(ethylene glycol) moiety. For example, NH₂(CH₂CH₂0)nCH₂C(=0)OH(e.g., 8-amino-3,6-dioxaoctanoic acid (AEEAc) or11-amino-3,6,9-trioxaundecanoic acid) or an NHS ester thereof (e.g., anNHS ester of 8-amino-3,6-dioxaoctanoic acid or1l-amino-3,6,9-trioxaundecanoic acid), can be used. In some embodiments,the resulting compound is as follows (wherein the portion contributed bythe linker is shown in bold):

(SEQ ID NO: 49) NH₂(CH₂)_(5c) (═0)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-N¾ (SEQ ID NO: 50)NH₂(CH₂CH₂0)₂CH₂C(═0)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH₂

In some embodiments, a Lys residue is attached to the N- or C-terminusof SEQ ID NO: 37 via a linker comprising a non-peptide portion. Forexample, the linker can comprise an alkyl chain, oligo(ethylene glycol)chain, and/or cyclic ring system. In some embodiments, 8-AEEAc or an NHSester thereof is used, resulting (in the case of attachment of Lys atthe C-terminus) in the following compound (wherein the portioncontributed by 8-AEEAc is shown in bold):

(SEQ ID NO: 51) {circumflex over( )}ic-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr -NH-CH₂CH₂OCH ₂CH₂OCH ₂-C(═0)-Lys -NH₂

It will be appreciated that in SEQ ID NOs: 49 and 50, a —C(=0) moiety isattached to the adjacent Ile residue via a C—N bond, wherein the N ispart of the amino acid and is not shown. Similarly, in SEQ ID NO: 51. a—C(=0) moiety is attached to the adjacent Lys residue via a C—N bond,wherein the N is part of the amino acid and is not shown. It will alsobe appreciated that that in SEQ ID NO: 51 the N H moiety is attached tothe immediately N-terminal amino acid (Thr), via a C—N bond, wherein theC is the carbonyl carbon of the amino acid and is not shown.

The compounds of SEQ ID NOs: 47-51 can readily be modified at theprimary amine group to produce a reactive compstatin analog. Forexample, the compounds of SEQ ID NOs: 47-51 (or other compoundscomprising a primary or secondary amine and a compstatin analog moiety)can be reacted with 6-maleimidocaproic acid N-succinimidyl ester toproduce the following reactive compstatin analogs:

(SEQ ID NO: 52) {circumflex over( )}c-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-(C(═0)-(CH ₂)₅-Mal)-NH₂. (SEQ ID NO: 53) {circumflexover ( )}c-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly) ₅-Lys--(C(═0)-(CH ₂)₅-Mal)-NH₂. (SEQ ID NO: 54)Mal-(CH₂)₅-(C(═0)-NH(CH₂) ₅C(═0)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr -NH₂ (SEQ ID NO: 55)Mal-(CH₂)₅-(C(═0)NH(CH ₂CH₂0)₂CH₂C(═0)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr - NH₂ (SEQ ID NO: 56){circumflex over ( )}ic-Ile-Cys*-Val-(lMe)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr -NH-CH₂CH₂OCH ₂CH₂OCH ₂-C(═0)-Lys- (C(═0)-(CH ₂)₅-Mal)-NH₂

In another embodiment, a reactive compstatin analog is represented as:{circumflex over( )}c-Ile-Cys*-Val-(lMe)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-C(═0)-CH₂(OCH₂CH2)₂NH(C(=0)-(CH₂)₅-Mal)-NH₂(SEQ ID NO: 57).

In some embodiments variants of SEQ ID NOs: 38-57 may be used in which-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr- is replacedby an amino acid sequence comprising the amino acid sequence of anyother compstatin analog, e.g., of any of SEQ ID NOs 3-27 or 29-36, 37,37A, 38A, 39A, 40A, or 41A with the proviso that blocking moiet(ies)present at the N- and/or C-termini of a compstatin analog may be absent,replaced by a linker (which may comprise a blocking moiety), or attachedto a different N- or C-terminal amino acid present in the correspondingvariant(s).

Other bifunctional cross-linkers comprising a maleimide as a reactivemoiety and an NHS ester as an amine-reactive moiety of use in variousembodiments include, e.g., succinimidyl 4-(p-maleimidophenyl)butyrate(SMPB); succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(SMCC); N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). Addition of asulfonate to the NHS ring results in water-soluble analogs such assulfo-succinimidyl(4-iodoacetyl)-aminobenzoate (sulfo-SIAB),sulfo-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(sulfo-SMCC), sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate(sulfo-SMPB), sulfo-N-y-maleimidobutyryl-oxysuccinimide ester(sulfo-GMBS) etc., which can avoid the need for an organic solvent. Insome embodiments, a long chain version of any of the foregoing,comprising a spacer arm between the NHS ester moiety and the remainderof the molecule, is used. The spacer can comprise, e.g., an alkyl chain.An example issuccinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate].

In some embodiments, a bifunctional linker comprising an NHS ester (asan amine-reactive moiety) and an iodoacetyl group (reactive withsulfhydryl groups) is used. Such linkers include, e.g.,N-succinimidyl(4-iodoacetyl)-aminobenzoate (SIAB); succinimidyl6-[(iodoacetyl)-amino]hexanoate (SIAX); succinimidyl6-[6-(((iodoacetyl)amino)-hexanoyl) amino]hexanoate (SIAXX);succinimidyl 4-((iodoacetyl)amino)methyl)-cyclohexane-1-carboxylate(SIAC); succinimidyl6-((((4-(iodoacetyl)amino)methyl-cyclohexane-1-carbonyl)amino)hexanoate(SIACX);

In some embodiments, a bifunctional linker comprising an NHS ester (asan amine-reactive moiety) and a pyridy disulfide group (as acell-reactive moiety reactive with sulfhydryl groups) is used. Examplesinclude N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP);succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (SMPT) andversions comprising a sulfonate on the NHS ring and/or a spacercomprising an alkyl chain between the NHS ester moiety and the rest ofthe molecule (e.g., succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate) (LC-SPDP). Variations ofsuch linkers that include additional or different moieties could beused. For example, a longer or shorter alkyl chain could be used in aspacer, or an oligo(ethylene glycol) moiety instead of an alkyl chain.

Any of the reactive compstatin analog moieties may be reacted with acompound comprising a CPM and a compatible reactive functional group.For example, a CPP or other CPM may be modified to incorporate areactive functional group capable of reacting with the reactivefunctional group of a reactive compstatin analog or of a linker to forma compound of formula A-L-M.

In general, a cell-penetrating compstatin analog can be synthesizedusing a variety of approaches. Compounds that comprise a reactivefunctional group and a linker can often be purchased as preformedbuilding blocks. For example, 6-malemeidocaproic acid and6-maleimidocaproic acid N-hydroxysuccinimide ester can be purchased fromvarious suppliers. Alternately, such compounds can be synthesized usingmethods known in the art. See, e.g., Keller O, Rudinger J. Helv ChimActa. 58(2):531-41, 1975 and Hashida S, et al, J Appl Biochem.,6(1-2):56-63, 1984. See also, Hermanson, G. supra, and referencestherein, for discussion of methods and reagents of use for synthesizingconjugates.

In some embodiments, an amino acid having a linker attached to a sidechain is used in the synthesis of a linear peptide. The linear peptidecan be synthesized using standard methods for peptide synthesis known inthe art, e.g., standard solid-phase peptide synthesis. The linearpeptide is then cyclized (e.g., by oxidation of the Cys residues to forman intramolecular disulfide). The cyclic compound may then be reactedwith a linker attached to a CPM. In other embodiments, a moietycomprising a CPM is reacted with a linear compstatin analog prior tocyclization thereof. In general, reactive functional groups can beappropriately protected to avoid undesired reaction with each otherduring synthesis of a cell-reactive compstatin analog. A reactivefunctional group, any of the amino acid side chains, and/or either orboth termini of a peptide may be protected during the reaction andsubsequently deprotected. For example, SH groups of Cys residues and/orSH-reactive moieties such as maleimides can be protected until aftercyclization to avoid reaction between them. The reaction conditions areselected based at least in part on the requirements of the particularreactive functional group(s) to achieve reasonable yield in a reasonabletime period. Temperature, pH, and the concentration of the reagents canbe adjusted to achieve the desired extent or rate of reaction. See,e.g., Hermanson, supra. The desired product can be purified, e.g., toremove unreacted compound(s), linker(s), products other than the desiredcell-penetrating compstatin analog that may have been generated in thereaction, other substances present in the reaction mixture, etc.Compositions and methods for making the cell-penetrating compstatinanalogs, and intermediates in the synthesis, are aspects of theinvention.

In some aspects of the invention, a compound comprises acell-penetrating compstatin analog and a moiety such as a polyethyleneglycol (PEG) chain or other polymer(s) that, e.g., stabilizes thecompound, increases its lifetime in the body, increases its solubility,decreases its immunogenicity, and/or increases its resistance todegradation. Without limiting the invention in any way, such a moietymay be referred to herein as a “clearance reducing moiety” (CRM).

In certain embodiments a CPCA is stable in physiological conditions forat least 24 hours or more. In certain embodiments a CPCA is stable inmammalian, e.g., primate, e.g., human or non-human primate (e.g.,monkey) blood, plasma, or serum for at least 24 hours. In variousembodiments at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,or more, of the CPCA molecules remains intact upon incubation inphysiological conditions for 24 hours, 48 hours, 72 hours, 96 hours, 120hours, 144 hours, 168 hours, or more. In various embodiments at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of the CPCAmolecules remains intact upon incubation in blood, plasma, or serum at37 degrees C. for 48 hours, 72 hours, 96 hours, 120 hours, 144 hours,168 hours, or more. Incubation may be performed using a CPCA at aconcentration of between 1 microgram/ml to about 100 mg/ml in variousembodiments. Samples may be analyzed at various time points. Size orintactness may be assessed using, e.g., chromatography (e.g., HPLC),mass spectrometry, Western blot, or any other suitable method.

In some embodiments, a CPCA has a molar activity of at least about 10%,20%, 30%, e.g., between 30% and 40%, between 30% and 50%, between 30%and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%,or more, of the activity of a corresponding compstatin analog having thesame amino acid sequence (and, if applicable, one or more blockingmoiet(ies)) but not comprising a CPM. In some embodiments wherein acompound comprises multiple compstatin analog moieties, the molaractivity of the long-acting compstatin analog is at least about 10%,20%, or 30%, e.g., between 30% and 40%, between 30% and 50%, between 30%and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%,or more, of the sum of the activities of said compstatin analogmoieties. In some embodiments, a polyethylene glycol (PEG) comprises a(CH₂CH₂0)_(n) moiety having a molecular weight of at least 500 daltons,e.g., an average molecular weight of between about 500; 1,000; 1,500;2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000;80,000; 90,000; and 100,000 daltons. In some embodiments the averagemolecular weight of a PEG is at least 20,000 daltons, up to about100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons.“Average molecular weight” refers to the number average molecularweight. In some embodiments, the polydispersity D of a (CH₂CH₂0)n moietyis between 1.0005 and 1.50, e.g., between 1.005 and 1.10, 1.15, 1.20,1.25, 1.30, 1.40, or 1.50, or any value between 1.0005 and 1.50.

In some embodiments, a (CH₂CH₂0)n moiety is monodisperse and thepolydispersity of a (CH₂CH₂0)n moiety is 1.0. Such monodisperse(CH₂CH₂0)n moieties are known in the art and are commercially availablefrom Quanta BioDesign (Powell, Ohio), and include, by way of nonlimitingexample, monodisperse moieties where n is 2, 4, 6, 8, 12, 16, 20, or 24.

In some embodiments, a compound comprises multiple (CH₂CH₂0)_(n)moieties wherein the total molecular weight of said (CH₂CH₂0)_(n)moieties is between about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000;50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In someembodiments the average total molecular weight of the compound or(CH₂CH₂0)_(n) moieties is at least 20,000 daltons, up to about 100,000;120,000; 140,000; 160,000; 180,000; or 200,000 daltons. In someembodiments, the compound comprises multiple (CH₂CH₂0)_(n) moietieshaving defined lengths, e.g., n=4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, or 30 or more. In some embodiments, the compound comprises asufficient number of (CH₂CH₂0)_(n) moieties having defined lengths toresult in a total molecular weight of said (CH₂CH₂0)_(n) moieties ofbetween about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000;60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodimentsthe average total molecular weight of the compound or (CH₂CH₂0)_(n)moieties is at least 20,000 daltons, up to about 100,000; 120,000;140,000; 160,000; 180,000; or 200,000 daltons. In some embodiments n isbetween about 30 and about 3000. In some embodiments a CPCA is attachedat each end of a linear PEG. A bifunctional PEG having a reactivefunctional group at each end of the chain may be used. In someembodiments the reactive functional groups are identical while in someembodiments different reactive functional groups are present at eachend. In some embodiments, multiple (CH₂CH₂0)_(n) moieties are providedas a branched structure. The branches may be attached to a linearpolymer backbone (e.g., as a comb-shaped structure) or may emanate fromone or more central core groups, e.g., as a star structure. In someembodiments, a branched molecule has 3 to 10 (CH₂CH₂0)_(n) chains. Insome embodiments, a branched molecule has 4 to 8 (CH₂CH₂0)_(n) chains.In some embodiments, a branched molecule has 10, 9, 8, 7, 6, 5, 4, or 3(CH₂CH₂0)_(n) chains. In some embodiments, a star-shaped molecule has10-100, 10-50, 10-30, or 10-20 (CH₂CH₂0)_(n) chains emanating from acentral core group. In some embodiments a compound thus may comprise,e.g., 3-10 CPCA moieties, e.g., 4-8 CPCA moities, each attached to a(CH₂(¾ 0)_(η) chain via a functional group at the end of the chain. Insome embodiments a compound may comprise, e.g., 10-100 CPCA moieties,each attached to a (CH₂(¾ 0)_(η) chain via a functional group at the endof the chain. In some embodiments, branches (sometimes referred to as“arms”) of a branched or star-shaped PEG contain about the same numberof (CH₂CH₂O) moieties. In some embodiments, at least some of the branchlengths may differ. It will be understood that in some embodiments oneor more (CH₂CI_(3/4 0)η) chains does not have a CPCA attached thereto.In some embodiments at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% of the chains has a CPCA attached thereto. In someembodiments any such compound may comprise both CPCA and compstatinanalog moieties without a CPM. At least some of the CPCA are attached toa PEG via a linker sufficiently labile to allow release of the CPCA,e.g., under physiological conditions in vitro, or in vivo.

In genera and compounds depicted herein, a polyethylene glycol moiety isdrawn with the oxygen atom on the right side of the repeating unit orthe left side of the repeating unit. In cases where only one orientationis drawn, the present invention encompasses both orientations (i.e.,(CH₂CH₂ _(0)n) and (OCH₂CH₂ _()n)) of polyethylene glycol moieties for agiven compound or genus, or in cases where a compound or genus containsmultiple polyethylene glycol moieties, all combinations of orientationsare encompasses by the present disclosure.

Formulas of some exemplary monofunctional PEGs comprising a reactivefunctional group are illustrated below. For illustrative purposes,formulas in which the reactive functional group(s) comprise an NHS esterare depicted, but other reactive functional groups could be used, e.g.,as described above. In some embodiments, the (CH₂CH₂ ₀₎ _(n) aredepicted as terminating at the left end with a methoxy group (OCH₃) butit will be understood that the chains depicted below and elsewhereherein may terminate with a different OR moiety (e.g., an aliphaticgroup, an alkyl group, a lower alkyl group, or any other suitable PEGend group) or an OH group. It will also be appreciated that moietiesother than those depicted may connect the (CH₂CH₂ ₀₎ _(n) moieties withthe NHS group in various embodiments.

In some embodiments, a monofunctional PEG is of formula A:

wherein “Reactive functional group” and n are as defined above anddescribed in classes and subclasses herein;

-   R¹ is hydrogen, aliphatic, or any suitable end group; and-   T is a covalent bond or a C₁₋₁₂ straight or branched, hydrocarbon    chain wherein one or more carbon units of T are optionally and    independently replaced by -0-, —S—, —N(R^(X))—, —C(0)-, —C(0)0-,    -0C(0)-, —N(R^(x))C(0)-, —C(0)N(R^(x))—, —S(0)-, —S(0)₂—,    —N(R^(x))S0₂—, or —S0₂N(R^(x))—; and-   each R^(x) is independently hydrogen or Ci_6 aliphatic.

Exemplary monofunctional PEGs of formula A include:

In Formula I, the moiety comprising the reactive functional group hasthe general structure —CO—(CH₂)_(m)—COO—NHS, where m=2. In someembodiments, a monofunctional PEGs has the structure of Formula I, wherem is between 1 and 10, e.g., between 1 and 5. For example, in someembodiments m is 3, as shown below:

In Formula II, the moiety comprising the reactive functional group hasthe general structure —(CH₂)_(m)—COO—NHS, where m=1. In some embodimentsa monofunctional PEG has the structure of Formula II, where m is between1 and 10 (e.g., wherein m is 5 as shown in Formula III below), orwherein m is 0 (as shown below in Formula Ilia).

In some embodiments a bifunctional linear PEG comprises a moietycomprising a reactive functional group at each of its ends. The reactivefunctional groups may be the same (homobifunctional) or different(heterobifunctional). In some embodiments the structure of abifunctional PEG may be symmetric, wherein the same moiety is used toconnect the reactive functional group to oxygen atoms at each end of the—(CH₂CH₂0)_(n) chain. In some embodiments different moieties are used toconnect the two reactive functional groups to the PEG portion of themolecule. The structures of exemplary bifunctional PEGs are depictedbelow. For illustrative purposes, formulas in which the reactivefunctional group(s) comprise an NHS ester are depicted, but otherreactive functional groups could be used.

In some embodiments, a bifunctional linear PEG is of formula B:

wherein each T and “Reactive functional group” is independently asdefined above and described in classes and subclasses herein, and n isas defined above and described in classes and subclasses herein.

Exemplary Afunctional PEGs of formula B include:

In Formula IV, the moiety comprising the reactive functional group hasthe general structure —(CH₂)_(m)—COO—NHS, where m=1. In someembodiments, a Afunctional PEG has the structure of Formula IV, where mis between 1 and 10, e.g., between 1 and 5. In certain embodiments m is0, e.g., embodiments the moiety comprising the reactive functional grouphas the general structure —COO—NHS. For example, in some embodiments abifunctional PEG has the structure of Formula IVa, as shown below:

In Formula V, the moiety comprising the reactive functional group hasthe general structure —CO—(CH₂)_(m)—COO—NHS, where m=2. In someembodiments, a bifunctional PEGs has the structure of Formula V, where mis between 1 and 10, e.g., between 1 and 5. In certain embodiments, forexample, m is 2, as shown below:

In some embodiments, the present invention provides a compstatin analogconjugated to a polymer. In some embodiments the compstatin analog is acell-penetrating compstatin analog. In certain embodiments, the presentinvention provides compstatin analog conjugates of PEG-containingcompounds and genera depicted herein, wherein in some embodiments thecompstatin analog is a CPCA. In some embodiments, a functional group(for example, an amine, hydroxyl, or thiol group) on a compstatin analogis reacted with a PEG-containing compound having a “reactive functionalgroup” as described herein, to generate such conjugates. By way ofexample, Formulae III and IV, respectively, can form compstatin analogconjugates having the structure:

wherein,

represents the attachment point of an amine group on a compstatinanalog, which may be a cell-penetrating compstatin analog. It should beunderstood that anywhere in formulas and genera herein, “COMPSTATINANALOG” may represent a cell-penetrating compstatin analog in someembodiments. In certain embodiments, an amine group is a lysine sidechain group. It will be appreciated that corresponding conjugates can beformed with any of the PEG-containing compounds and genera depictedherein, depending on the choice of reactive functional group and/orcompstatin functional group. For example, Formulae IVa and Va,respectively, can form compstatin analog conjugates having the followingstructures

The term “bifunctional” or “bifunctionalized” is sometimes used hereinto refer to a compound comprising two compstatin analog moieties linkedto a CRM. Such compounds may be designated with the letter “BF”. In someembodiments a bifunctionalized compound is symmetrical. In someembodiments the linkages between the CRM and each of the compstatinanalog moieties of a bifunctionalized compound are the same.

In some embodiments, a branched, comb, or star-shaped PEG comprises amoiety comprising a reactive functional group at the end of each ofmultiple —(CH₂CH₂0)_(n) chains. The reactive functional groups may bethe same or there may be at least two different groups. In someembodiments, a branched, comb, or star-shaped PEG is of the followingformulae:

wherein each R² is independently a “Reactive functional group” or R¹,and each T, n, and “Reactive functional group” is independently asdefined above and described in classes and subclasses herein. Thestructure of exemplary branched PEGs (having 8 arms, or branches)comprising NHS moieties as reactive functional groups is depicted below:

The structure of exemplary branched PEGs (having 4 arms, or branches)comprising NHS moieties as reactive functional groups is depicted below:

The number of branches emanating from the backbone may be varied. Forexample, the number 4 in the above formulae VI and VII may be changed toany other integer between 0 and 10 in various embodiments. In certainembodiments, one or more branches does not contain a reactive functiongroup and the branch terminates with a —CH₂CH₂OH or —CH₂CH₂OR group, asdescribed above.

In some embodiments a branched PEG has the structure of Formula VII,VIII, or IX (or variants thereof having different numbers of branches)with the proviso that x is

In some embodiments a branched PEG has the structure of Formula VII,VIII, or IX (or variants thereof having different numbers of branches)with the proviso that x is

Of course the methylene (CH2) group in the above x moiety may insteadcomprise a longer alkyl chain (CH₂)_(m), where m is up to 2, 3, 4, 5, 6,8, 10, 20, or 30, or may comprise one or more other moieties describedherein.

In some embodiments, exemplary branched PEGs having NHS or maleimdereactive groups are depicted below:

In some embodiments, a variant of Formula X or XI are used, wherein 3 oreach of the 4 branches comprise a reactive functional group.

Still other examples of PEGs may be represented as follows:

As noted above, it will be appreciated that, as described herein, invarious embodiments any of a variety of moieties may be incorporatedbetween the peptide component and (CH₂CH₂0)_(n)-R moiety of acell-penetrating or long-acting compstatin analog, such as an linearalkyl, ester, amide, aromatic ring (e.g., a substituted or unsubstitutedphenyl), a substituted or unsubstituted cycloalkyl structure, orcombinations thereof. In some embodiments such moiet(ies) may render thecompound more susceptible to hydrolysis, which may release the peptideportion of the compound from the CRM. In some embodiments, such releasemay enhance the in vivo tissue penetration and/or activity of thecompound. In some embodiments such release releases a CPCA, which maythen be able to be internalized by cells. In some embodiments hydrolysisis general (e.g., acid-base) hydrolysis. In some embodiments hydrolysisis enzyme-catalyzed, e.g., esterase-catalyzed. Of course both types ofhydrolysis may occur. Examples of PEGs comprising one or more suchmoieties and an NHS ester as a reactive functional group are as follows:

In some embodiments a branched (multi-arm) PEG or star-shaped PEGcomprises a pentaerythritol core, hexaglycerin core, ortripentaerythritol core. It will be understood that the branches may notall emanate from a single point in certain embodiments.

Monofunctional, bifunctional, branched, and other PEGs comprising one ormore reactive functional groups may, in some embodiments, be obtainedfrom, e.g., NOF America Corp. White Plains, N.Y. or BOC Sciences 45-16Ramsey Road Shirley, NY 11967, USA, among others, or may be preparedusing methods known in the art.

In some embodiments, the present invention provides a compstatin analogconjugated with a polymer, wherein the polymer is other than PEG. Insome embodiments, a polymer is a polyoxazoline (POZ). Exemplary mono-and poly-functionalized polyoxazoline derivatives for directconjugation, or for conjugation via a linker, are depicted below:

Z-T-[N(COR^(X))CH₂CH₂]_(″)-T-R¹;

R¹—{[N(CO-T-Z)CH₂CH₂]_(m)—[N(COR^(X))CH₂CH₂]_(n)}^(a)-T-R¹;

R¹—{[N(CO-T-Z¹)CH₂CH₂]_(p)—[N(COR^(X))CH₂CH₂]_(n)—[N(CO-T-Z²)CH₂CH₂]_(m)}^(a)-T-R¹;

R¹—{[N(CO-T-Z¹)CH₂CH₂]_(p)—[N(COR^(X))CH₂CH₂]_(n)—[N(CO-T-Z²)CH₂CH₂]_(m)}^(a)-T-Z;

R¹—[N(COR^(X))CH₂CH₂]_(n)-T-B(—R^(X))(-T-Z)-T-[N(COR^(X))CH₂CH₂]_(m)—R¹;

wherein:

-   -   each of Z, Z¹ and Z² is independently a reactive functional        group as defined above and described in classes and subclasses        herein;    -   each of T, R^(x), and R¹ is independently as defined above and        described in classes and subclasses herein;    -   each of m, n, and p is independently an integer 0-1000, with the        limitation that the sum of m, n, and p for each formula is not        0;    -   a is “ran,” which indicates a random copolymer, or “block,”        which indicates a block copolymer;    -   B is a branching moiety that is linked with or without a linker        to the other parts of the polymer.        Other examples of functionalized polyoxazoline derivatives for        conjugation are extensively described in the art, including but        not limited to those described in PCT Patent Application        Publication Nos. WO/2010/006282, WO/2009/089542, WO/2009/043027        and WO/2008/106186, the entirety of each of which is hereby        incorporated by reference.

Exemplary compstatin analog conjugates with polyoxazoline polymers aredepicted below:

wherein each variable is independently as defined above and described inclasses and subclasses herein.

In some embodiments, the present invention provides a compstatin analogconjugated with a polymer, wherein the compstatin analog is connected tothe polymer via one or more linkers. In some embodiments, a polymer isselected from PEG-containing compounds and genera described above and inclasses and subclasses herein. In some embodiments, the presentinvention provides compstatin analog conjugates of PEG-containingcompounds and genera depicted herein, wherein the compstatin analog isconnected to the PEG-containing moieties via one or more linkers. Mono-and poly-functional PEGs that comprise one or more reactive functionalgroups for conjugation are defined above and described in classes andsubclasses herein, including but not limited to those of formula A, I,la, II, III, Ilia, B, IV, IVa, V, Va, C, D, E, F, G, H, VI, VII, VIII,IX, X, XI, XII, XIII, XIV, XV, or XVI.

Suitable linkers for connecting a compstatin analog and a polymer moietysuch as PEG or polyoxazoline are extensively described above and inclasses and subclasses herein. In some embodiments, a linker hasmultiple functional groups, wherein one functional group is connected toa compstatin analog and another is connected to a polymer moiety. Insome embodiments, a linker is a bifunctional compound. In someembodiments, a linker has the structure of NH₂(CH₂CH₂0)nCH₂C(=0)OH,wherein n is 1 to 1000. In some embodiments, a linker is8-amino-3,6-dioxaoctanoic acid (AEEAc). In some embodiments, a linker isactivated for conjugation with a polymer moiety or a functional group ofa compstatin analog. For example, in some embodiments, the carboxylgroup of AEEAc is activated before conjugation with the amine group ofthe side chain of a lysine group.

In some embodiments, a suitable functional group (for example, an amine,hydroxyl, thiol, or carboxylic acid group) on a compstatin analog isused for conjugation with a polymer moiety, either directly or via alinker. In some embodiments, a compstatin analog is conjugated throughan amine group to a PEG moiety via a linker. In some embodiments, anamine group is the a-amino group of an amino acid residue. In someembodiments, an amine group is the amine group of the lysine side chain.In some embodiments, a compstatin analog is conjugated to a PEG moietythrough the amino group of a lysine side chain (e-amino group) via alinker having the structure of NH₂(CH₂CH₂0)nCH₂C(=0)OH, wherein n is 1to 1000. In some embodiments, a compstatin analog is conjugated to thePEG moiety through the amino group of a lysine side chain via an AEEAclinker. In some embodiments, the NH₂(CH₂CH₂0)nCH₂C(=0)OH linkerintroduces a —NH(CH₂CH₂0)nCH₂C(=0)- moiety on a compstatin lysine sidechain after conjugation. In some embodiments, the AEEAc linkerintroduces a —NH(CH₂CH₂0)₂CH₂C(=0)- moiety on a compstatin lysine sidechain after conjugation.

In some embodiments, a compstatin analog is conjugated to a polymermoiety via a linker, wherein the linker comprises an AEEAc moiety and anamino acid residue. In some embodiments, a compstatin analog isconjugated to a polymer moiety via a linker, wherein the linkercomprises an AEEAc moiety and a lysine residue. In some embodiments, apolymer is PEG. In some embodiments, the C-terminus of a compstatinanalog is connected to the amino group of AEEAc, and the C-terminus ofAEEAc is connected to a lysine residue. In some embodiments, theC-terminus of a compstatin analog is connected to the amino group ofAEEAc, and the C-terminus of AEEAc is connected to the a-amino group ofa lysine residue. In some embodiments, the C-terminus of a compstatinanalog is connected to the amino group of AEEAc, the C-terminus of AEEAcis connected to the a-amino group of the lysine residue, and a polymermoiety, such as a PEG moiety, is conjugated through the e-amino group ofsaid lysine residue. In some embodiments, the C-terminus of the lysineresidue is modified. In some embodiments, the C-terminus; of the lysineresidue is modified by amidation. In some embodiments, the N-terminus ofa compstatin analog is modified. In some embodiments, the N-terminus ofa compstatin analog is acetylated.

Exemplary conjugates comprising an AEEAc linker and polymer are depictedbelow, wherein

r_(e)pr_(e)s_(en)t_(s) the attachment point of an amine |group on acompstatin analog,

r_(e)pr_(e)s_(en)t_(s) a compstatin analog attaching through itsC-terminus, and wherein each of the other variables is independently asdefined above and described in classes and subclasses herewith. In someembodiments, an amine group is the amino group of a 1 sine side chain.

In certain embodiments a compstatin analog may be represented asM-AEEAc-Lys-5.2, wherein B₂ is a blocking moiety, e.g., NH₂, Mrepresents any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A, withthe proviso that the C-terminal amino acid of any of SEQ ID NOs: 3-36,37, 37A, 38A, 39A, 40A, or 41A is linked via a peptide bond toAEEAc-Lys-i?2. The NHS moiety of a monofunctional or multifunctional(e.g., bifunctional) PEG reacts with the free amine of the lysine sidechain to generate a monofunctionalized (one compstatin analog moiety) ormultifunctionalized (multiple compstatin analog moieties) long-actingcompstatin analog. In various embodiments any amino acid comprising aside chain that comprises a reactive functional group may be usedinstead of Lys (or in addition to Lys). A monofunctional ormultifunctional PEG comprising a suitable reactive functional group maybe reacted with such side chain in a manner analogous to the reaction ofNHS-ester activated PEGs with Lys.

With regard to any of the above formulae and structures, it is to beunderstood that embodiments in which the compstatin analog componentcomprises any compstatin analog described herein, e.g., any compstatinanalog of SEQ ID NOs; 3-36, 37, 37A, 38A, 39A, 40A, 41A, are expresslydisclosed. For example, and without limitation, a compstatin analog maycomprise the amino acid sequence of SEQ ID NO: 28.

In some aspects, the present invention relates to use of click chemistryin connection with compstatin analogs. “Click chemistry” is well knownin the art and is useful in some aspects of the present invention. Clickchemistry embodies, in certain embodiments, versatile cycloadditionreactions between azides and alkynes that enable a number of usefulapplications. Methods of carrying out click chemistry are known in theart, and are described by Kolb, H. C.; Sharpless, K. B., Drug Disc.Today, 2003, 1128-1137; Moses, J. E.; Moorhouse, A. D.; Chem. Soc. Rev.,2007, 1249-1262; the entire contents of each are hereby incorporated byreference. Click chemistry is a popular method of bioconjugation due toits high reactivity and selectivity, even in biological media. See Kolb,H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40,2004-2021; and Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.;Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. Inaddition, currently available recombinant techniques and syntheticmethods permit the introduction of azides and alkyne-bearingnon-canonical amino acids into peptides, proteins, cells, viruses,bacteria, and other biological entities that consist of or displayproteins. See Link, A. J.; Vink, M. K. S.; Tirrell, D. A. J. Am. Chem.Soc. 2004, 126, 10598-10602; Deiters, A.; Cropp, T. A.; Mukherji, M.;Chin, J. W.; Anderson, C; Schultz, P. G. J. Am. Chem. Soc. 2003, 125,11782-11783.

As used herein, the term “click chemistry group” is sometimes used torefer\ to a reactive functional group capable of participating in aclick chemistry reaction with an appropriate second reactive functionalgroup, which second reactive functional group is also a click chemistrygroup. The first and second click chemistry groups, or entities (e.g.,molecules) comprising such groups, may be referred to as complementary.First and second entities, e.g., molecules, that comprise complementaryclick chemistry groups may be referred to as click chemistry partners.An entity or molecule comprising a click chemistry group may be referredto as “click-functionalized”. A bond formed by reaction of complementaryclick chemistry partners may be referred to as a “click chemistry bond”.

In some embodiments, the present invention provides click-functionalizedcompstatin analogs for, e.g., conjugation to a complementary moiety on apartner molecule or biomolecule. In some embodiments, a complementarypartner molecule or biomolecule is a polymer, peptide, protein, or amolecule that functions as a clearance-reducing moiety. In someembodiments, the “click-functionalized” moiety is an alkyne or an alkynederivative which is capable of undergoing [3+2] cycloaddition reactionswith complementary azide-bearing molecules and biomolecules. In anotherembodiment, the “click-functionalized” functionality is an azide or anazide derivative which is capable of undergoing [3+2]cycloadditionreactions with complementary alkyne-bearing molecules and biomolecules(i.e. click chemistry).

In some embodiments, a click-functionalized compstatin analog bears anazide group on any side chain group of the compstatin analog. In someembodiments, a click-functionalized compstatin analog bears an azidegroup on a lysine side chain group.

In some embodiments, a click-functionalized compstatin analog bears analkyne group on any side chain group of the compstatin analog. In someembodiments, a click-functionalized compstatin analog bears an alkynegroup on a lysine side chain group.

In some embodiments, the present invention provides compstatinconjugates comprising a compstatin analog, a molecule that functions asa clearance-reducing moiety, and a triazole linker. In some embodiments,a triazole linker is the result of click conjugation chemistry between acompstatin conjugate and a molecule that functions as aclearance-reducing moiety. In some embodiments the CRM may be any CRMdisclosed herein. For example, the CRM may be a PEG, a polypeptide, or aPOZ.

In some embodiments, the present invention provides compstatinconjugates comprising a compstatin analog, a PEG moiety, and a triazolelinker. In some embodiments, a triazole linker is the result of clickconjugation chemistry between a compstatin conjugate and a PEG moiety.

In some embodiments, the present invention provides compstatinconjugates comprising a compstatin analog, a polyoxazoline moiety, and atriazole linker. In some embodiments, a triazole linker is the result ofclick conjugation chemistry between a compstatin conjugate and apolyoxazoline moiety.

In some embodiments, click chemistry between a compstatin analog andanother moiety is transition metal catalyzed. Copper-containingmolecules which catalyze the “click” reaction include, but are notlimited to, copper wire, copper bromide (CuBr), copper chloride (CuCl),copper sulfate (CuS0₄). copper sulfate pentahydrate (CuS0₄»5H₂0), copperacetate (Cu₂(Ac0₄). copper iodide (Cul), [Cu(MeCN)₄](OTf),[Cu(MeCN)₄](PF₆), colloidal copper sources, and immobilized coppersources. In some embodiments other metals, such as ruthenium. Reducingagents as well as organic and inorganic metal-binding ligands can beused in conjunction with metal catalysts and include, but are notlimited to, sodium ascorbate, tris(triazolyl)amine ligands,tris(carboxyethyl)phosphine (TCEP), sulfonated bathophenanthrolineligands, and benzimidazole-based ligands.

In some embodiments, compstatin analogs are conjugated to othermoieties, e.g., CPMs, CRMs, using metal free click chemistry (also knownas copper free click chemistry) to give a metal free composition orconjugates. In contrast to standard click chemistry, also known ascopper assisted click chemistry (CuACC), metal free click chemistryoccurs between either a strained, cyclic alkyne or an alkyne precursorsuch as an oxanorbornadiene, and an azide group. As the name implies, nometal catalyst is necessary for the reaction to occur. Examples of suchchemistries include reactions involving cyclooctyne derivatives(Codelli, et. al. J. Am. Chem. Soc, 2008, 130, 11486-11493; Jewett, et.al. J. Am. Chem. Soc., 2010, 132, 3688-3690; Ning, et. al. Angew. Chem.Int. Ed., 2008, 47, 2253-2255), difluoro-oxanorbornene derivatives (vanBerkel, et. al. ChemBioChem, 2007, 8, 1504-1508), or nitrile oxidederivatives (Lutz, et. al. Macromolecules, 2009, 42, 5411-5413). Incertain embodiments a metal-free click chemistry reaction is ametal-free [3+2] cycloaddition reaction, Diels-Alder reaction, orthiol-alkene radical addition reaction. Exemplary click chemistryreactions and click chemistry groups are described in, e.g., JoergLahann, Click Chemistry for Biotechnology and Materials Science, 2009,John Wiley & Sons Ltd, ISBN 978-0-470-69970-6; Becer, Hoogenboom, andSchubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition,Angewandte Chemie International Edition (2009) 48: 4900-4908. In certainembodiments a click chemistry group comprises a diarylcyclooctyne.

Certain examples of metal free click chemistry are shown in the schemebelow.

Certain metal-free click moieties are known in the literature. Examplesinclude 4-dibenzocyclooctynol (DIBO)

(from N ing et. al; Angew Chem Int Ed, 2008, 47, 2253); difluorinatedcyclooctynes (DIFO or DFO)

(from Codelli, et. al; J. Am. Chem. Soc. 2008, 130, 11486-11493);biarylazacyclooctynone (BARAC)

(from Jewett et. al; J. Am. Chem. Soc. 2010, 132, 3688); orbicyclononyne (BCN)

(From Dommerholt, et. al; Angew Chem Int Ed, 2010, 49, 9422-9425) ordibenzylcyclooctyne (DBCO)

A reaction scheme involving reaction of DBCO and an azide is shownbelow:

In the above scheme, in various embodiments, A may comprise or consistof a compstatin analog moiety and B may comprise or consist of a CPM orCRM, e.g., a polymer, such as a PEG or POZ a polypeptide, or B maycomprise or consist of a compstatin analog moiety and A may comprise orconsist of a CPM or CRM, e.g., a polymer, such as a PEG or POZ or apolypeptide.

In some embodiments, the “metal free click-functionalized” moiety is anacetylene or an acetylene derivative which is capable of undergoing[3+2] cycloaddition reactions with complementary azide-bearing moleculesand biomolecules without the use of a metal catalyst.

In some embodiments, the R and R′ groups of the metal-free clickchemistry reagents are a compstatin analog or any molecule describedherein to which a compstatin analog may be conjugated. In someembodiments, such compstatin analogs bear a click-functionalized moietyon a lysine side chain. In some embodiments, such compstatin analogs areconnected to a click-functionalized moiety via a linker. In someembodiments, such compstatin analogs are connected to aclick-functionalized moiety via AEEAc.

In some embodiments, a click chemistry reagent comprises DBCO. Exemplaryreagents and exemplary uses thereof are set forth below:

DBCO-Acid. In some embodiments a DBCO-Acid may be used to react with anamine-containing moiety.

DBCO-NHS ester (above) or DBCO-sulfo-NHS ester (below) may be used toincorporate a DBCO functionality into an amine-containing molecule, suchas a compstatin analog or a polypeptide comprising a lysine residue.

DBCO-PEG 4-NHS ester. In some embodiments such reagent is useful forintroducing a DBCO moiety by reaction with an available aminefunctionality. In some aspects, the presence of a PEG chain as ahydrophiiic spacer may be useful to, e.g., increase solubility orprovide flexibility.

DBCO-Arnine. In some embodiments a click chemistry reagent comprises acarbonyl/carboxyf reactive dibenzylcyclooctyne, which may react withacids, active esters and/or aldehydes.

In certain embodiments a click chemistry reaction involves a cyclooctynedepicted below:

In certain embodiments click chemistry reactions comprise reactionsbetween nitrones and cyclooctynes (see, e.g., Ning, Xinghai; Temming,Rinske P.; Dommerholt, Jan; Guo, Jun; Ania, Daniel B.; Debets, MarjokeF.; Wolfert, Margreet A.; Boons, Geert-Jan et al. (2010). “ProteinModification by Strain-Promoted Alkyne-Nitrone Cycloaddition”.Angewandte Chemie International Edition 49 (17): 3065), oxime/hydrazoneformation from aldehydes and ketones, tetrazine ligations (see, e.g.,Blackman, Melissa L.; Royzen, Maksim; Fox, Joseph M. (2008). “TheTetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demandDiels-Alder Reactivity”. Journal of the American Chemical Society 130(41): 13518-9), tetrazole ligations, the isonitrile-based click reaction(see, e.g., Stackmann, Henning; Neves, AndrA© A.; Stairs, Shaun;Brindle, Kevin M.; Leeper, Finian J. (2011). “Exploring isonitrile-basedclick chemistry for ligation with biomolecules”. Organic & BiomolecularChemistry 9 (21): 7303), and the quadricyclane ligation (see, e.g.,Sletten, Ellen M.; Bertozzi, Carolyn R. (2011). “A BioorthogonalQuadricyclane Ligation”. Journal of the American Chemical Society 133(44): 17570-3). In certain embodiments a click chemistry reaction is aStaudinger ligation (phosphine-azide).

Any compstatin analog may be modified to incorporate a click chemistrygroup in various embodiments. For example, a compstatin analogcomprising the sequence of any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A,40A, or 41A may be so modified. In some embodiments any such sequencefurther comprises a lysine residue or an AEEAc-Lys moiety, e.g., at theC-terminus. In some embodiments a click chemistry group is incorporatedafter peptide synthesis. For example, a Lys side chain may be reactedwith azido acetic acid in order to introduce an azide moiety as a clickchemistry group. In some embodiments a click chemistry group isincorporated after cyclization and, in some embodiments, after additionof a blocking moiety at the N- and/or C-terminus. In some embodiments aclick chemistry group is incorporated during peptide synthesis. Forexample, an amino acid comprising a side chain that comprises a clickchemistry group may be used in the synthesis of a compstatin analog. Avariety of such amino acids are commercially available from a number ofsources, e.g., AAPPTec (Louisville, Ky.), Jena Bioscience GmBH (Jena,Germany). In some aspects, methods of making a click chemistryfunctionalized compstatin analog are provided herein.

In some embodiments compositions comprising a compstatin analog and aclick chemistry reagent are used to install a click chemistry group,e.g., any click chemistry group known in the art, to a compstatinanalog. In some aspects, the composition may be incubated under suitableconditions (which may include providing a suitable catalyst, light(e.g., UV)) to functionalize the compstatin analog with a clickchemistry functionality. In some embodiments methods of making acell-penetrating compstatin analog are provided. In some embodiments themethods comprise mixing a compstatin analog comprising a first clickchemistry group with a CPM comprising a complementary click chemistrygroup under conditions suitable for a click chemistry reaction to occur.Additional steps may comprise purifying the resulting conjugate. In someembodiments purifying comprises removing at least some unreactedcomponents, e.g., with an appropriate scavenger.

In some embodiments a cell-penetrating compstatin analog may be usedtogether with a long-acting compstatin analog. For example, a method oftreatment may comprise administering a cell-penetrating compstatinanalog and a long-acting compstatin analog. Exemplary long-actingcompstatin analogs are set forth below and herein.

(SEQ ID NO: 58) (CH₂CH₂0)nC(═0)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr- NH₂) (SEQ ID NO: 59) {circumflex over( )}ic-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr -NH-CH₂CH₂OCH₂CH₂OCH₂-C(═0)-Lys-C (═0)-(CH ₂CH₂0)n -NH₂(SEQ ID NO: 60) {circumflex over( )}c-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-C(═0)-(CH₂CH₂0)n -NH₂. (SEQ ID NO: 61) {circumflex over( )}c-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly) ₅-Lys-C(═0)-(CH ₂CH₂0)n -NH₂ (SEQ ID NO: 62){circumflex over ( )}c-(CH ₂CH₂0)nC(═0)Lys-(Gly)5-Ile- Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr - JV¾) (SEQ ID NO: 63){circumflex over ( )}c-(CH ₂CH₂0)nC(═0)Lys-Ile- Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr - NH₂)

In SEQ ID NO: 58, the (CH₂CH₂0)n is coupled via an amide bond to theN-terminal amino acid. In SEQ ID NOs: 59-63, the (CH₂CH₂0)n moiety iscoupled via an amide bond to a Lys side chain; thus it will beunderstood that the NM at the C-terminus in SEQ ID NOs: 59, 60, and 61,represents amidation of the C-terminus of the peptide, and it will beunderstood that in SEQ ID NOs: 62 and 63, the A c at the N-terminusrepresents acetylation of the N-terminus of the peptide, as describedabove. It will also be appreciated by those of ordinary skill in the artthat a free end of a (CH₂CH₂0)_(n) moiety typically terminates with an(OR) where the underlined O represents the O atom in the terminal(CH₂CH₂0) group. (OR) is often a moiety such as a hydroxyl (OH) ormethoxy (—OCH₃) group though other groups (e.g., other alkoxy groups)could be used. Thus SEQ ID NO: 59, for example, may be represented as{circumflex over( )}c-Ile-Cys*-Val-(lMe)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH—CH₂CH₂OCH₂CH₂OCH₂—C(=0)-Lys-(C(=0)-(CH₂CH₂0)_(″)-R)—NH2(SEQ ID NO: 64) wherein R is, e.g., either H or CH₃ in the case of alinear PEG. In the case of a bifunctional, branched or star-shaped PEG,R represents the remainder of the molecule. Further, it will beunderstood that the moiety comprising the reactive functional group mayvary, as described herein (e.g., according to any of the formulasdescribed herein). For example, long-acting compstatin analogscomprising the same peptide sequence as SEQ ID NO: 64, in which themoiety comprising the reactive functional group comprises an esterand/or alkyl chain may be represented as follows

(SEQ ID NO: 65) {circumflex over( )}ic-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═0)-Lys-(C(═0)-(CH₂) m(CH₂CH₂0),,-R )-NH₂; (SEQ ID NO: 66) {circumflex over( )}ic-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═0)-Lys-(C(═0)-(CH ₂)_(m)-C(═0)-(CH₂CH₂0),,-R )-NH₂ (SEQ ID NO: 67) {circumflexover ( )}ic-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═0)-Lys-(C(═0)-(CH ₂)_(m)-C(═0)-(CH₂)_(j) (CH₂CH₂0)_(n)-R)-NH₂In SEQ ID NOs: 65-67 m may range from 1 up to about 2, 3, 4, 5, 6, 7, 8,10, 15, 20, or 30 in various embodiments, In SEQ ID NOs: 67j may rangefrom 1 up to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in variousembodiments.It will also be appreciated that, as described herein, in variousembodiments other moieties may be incorporated between the Lys-(C(=0)-and (CH2CH20)n-R, such as an amide, aromatic ring (e.g., a substitutedor unsubstituted phenyl), or a substituted or unsubstituted cycloalkylstructure.

In some embodiments variants of SEQ ID NOs: 58-67 are used in which-Ile-Cys*-Val-(lMe)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr- is replacedby an amino acid sequence comprising the amino acid sequence of anyother compstatin analog, e.g., of any of SEQ ID NOs 3-27 or 29-36, 37,37A, 38A, 39A, 40A, or 41A with the proviso that blocking moiet(ies)present at the N- and/or C-termini of a compstatin analog may be absent,replaced by a linker (which may comprise a blocking moiety), or attachedto a different N- or C-terminal amino acid present in the correspondingvariant(s).

Any compstatin analog, e.g., any compound comprising any of SEQ ID NOs:3-37, 37A, 38A, 39A, 40A, or 41A may, in various embodiments, can beattached via or near its N-terminal or C-terminal end (e.g., via a sidechain of an amino acid at or near its N-terminal or C-terminal aminoacid) directly or indirectly to any moiety comprising a CPM, e.g., aCPP, e.g., a CPP comprising or derived from a sequence listed in Table2.

Any compstatin analog, e.g., any compound comprising any of SEQ ID NOs:3-37, 37A, 38A, 39A, 40A, or 41A may, in various embodiments, can beattached via or near its N-terminal or C-terminal end (e.g., via a sidechain of an amino acid at or near its N-terminal or C-terminal aminoacid) directly or indirectly to any moiety comprising a reactivefunctional group, e.g., any compound of Formulae I-XVI or Formulae A-H.

In some embodiments, producing a cell-penetrating compstatin analogcomprises reacting a compstatin analog comprising a reactive functionalgroup with the N-terminal amine and/or C-terminal carboxyl group of apolypeptide comprising a CPP. In some embodiments, producing along-acting compstatin analog comprises reacting a compstatin analogcomprising an amine-reactive functional group with amino acids having aside chain comprising a primary amine (e.g., lysine) and/or with theN-terminal amine of a polypeptide comprising a CPP. In some embodiments,producing a cell-penetrating compstatin analog comprises reacting acompstatin analog comprising a carboxyl-reactive functional group withthe C-terminal carboxyl group of a polypeptide comprising a CPP. In someembodiments, producing a cell-penetrating compstatin analog comprisesreacting a compstatin analog comprising a sulfhydryl-reactive functionalgroup with one or more sulfhydryl groups of the polypeptide.

In some embodiments, at least one reactive functional group isintroduced into a polypeptide comprising a CPP. For example, in someembodiments at least one side chain of the polypeptide is modified toconvert a first reactive functional group to a different reactivefunctional group prior to reaction with the compstatin analog. In someembodiments a thiol is introduced. Several methods are available forintroducing thiols into biomolecules, including the reduction ofintrinsic disulfides, as well as the conversion of amine, aldehyde orcarboxylic acid groups to thiol groups. Disulfide crosslinks of cystinesin proteins can be reduced to cysteine residues by dithiothreitol (DTT),tris-(2-carboxyethyl)phosphine (TCEP), or tris-(2-cyanoethyl)phosphine.Amines can be indirectly thiolated by reaction with succinimidyl3-(2-pyridyldithio)propionate (SPDP) followed by reduction of the3-(2-pyridyldithio)propionyl conjugate with DTT or TCEP. Amines can beindirectly thiolated by reaction with succinimidyl acetylthioacetatefollowed by removal of the acetyl group with 50 mM hydroxylamine orhydrazine at near-neutral pH. Amines can be directly thiolated byreaction with 2-iminothiolane, which preserve the overall charge of themolecule and introduces a free thiol. Tryptophan residues in thiol-freeproteins can be oxidized to mercaptotryptophan residues, which can thenbe modified by iodoacetamides or maleimides. A polypeptide comprising aCPP and comprising one or more thiols may be reacted with a compstatinanalog comprising a maleimide group, such as {circumflex over( )}c-Ile-Cys*-Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-AEEAc-Lys-(C(=0)-(CH₂)₅-Mal)-NH₂to generate a cell-penetrating compstatin analog.

VI. Various Aspects

In some aspects, the invention provides a composition comprising a CPCAand at least one isolated cell. In some embodiments the cell comprises anucleic acid that encodes a primate C3. In some embodiments the cell isa primate cell, and the nucleic acid is endogenous to the cell (i.e., itis naturally part of the genome of the cell and was not introduced intothe cell or an ancestor of the cell by man). In some embodiments thenucleic acid is not endogenous to the cell but instead was introducedinto the cell or an ancestor of the cell by man. In some embodiments thenucleic acid that encodes a primate C3 is in a vector. In someembodiments the nucleic acid that encodes a primate C3 is integratedinto the genome. In some embodiments the nucleic acid that encodes aprimate C3 is operably linked to a heterologous promoter. In someembodiments the heterologous promoter is a regulatable promoter, e.g.,an inducible or repressible promoter. The cell may be of any cell typein various embodiments. In some embodiments the cell is of a type thatnaturally expresses a primate C3 under at least some conditions. In someembodiments the cell expresses a human C3 under at least someconditions. In some embodiments the cell is a human cell. In someembodiments the cell is maintained under conditions in which itexpresses C3. In some embodiments the composition comprises two or moredifferent types of cells, wherein a first cell type expresses C3 and asecond cell type expresses a C3a receptor (either naturally or as aresult of introduction of a heterologous nucleic acid encoding a C3areceptor). In some embodiments a cell in any of the compositions is anepithelial cell, e.g., an epithelial cell originating from any of theepithelial tissues mentioned above. In some embodiments a cell is arespiratory epithelial cell, retinal pigment epithelial (RPE) cell,endothelial cell, hepatocyte, immune system cell, e.g., lymphocyte (Bcell, T cell (e.g., CD4+ T cell, CD8+ T cell, Thl7 cell, Treg cell, NK Tcell), NK cell), granulocyte (e.g., neutrophil, eosinophil, basophil)mast cell, monocyte, or macrophage. In some embodiments a cell is anervous system cell, e.g., a neural cell or glial cell. In someembodiments a cell is a fully differentiated cell. In some embodiments acell is a precursor cell, e.g., a precursor of any of theafore-mentioned cell types. In the compositions comprising two or morecell types, the first and second cell types may be any of these celltypes in various embodiments. All combinations are encompassed. In someembodiments the composition comprises culture medium suitable forculturing the cells. In some embodiments the culture medium comprises anagent that stimulates C3 expression or intracellular C3 cleavage. Insome embodiments the culture medium comprises an agent that activates orinhibits one or more biological activities of one or more cell types inthe composition. In some embodiments the agent comprises a cytokine,growth factor, or agent that modulates expression or activity of acytokine or cytokine receptor. In some embodiments the culture mediumcomprises one, two, three or more cytokines and/or growth factors. Insome embodiments the culture medium comprises one, two, three or moreagents (e.g., small molecules, antibodies, polypeptides, or nucleicacids) that modulate (e.g., increase or decrease) expression or activityof a cytokine or cytokine receptor. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, MIP-1,MIP-2, MIP-3, MCP-1, MCP-2, MCP-3. In some embodiments a cytokine is aThl7-associated cytokine (e.g., IL-17, IL-23, or IL-26, or othercytokine produced by Thl7 cells or that activates Thl7 cells). In someembodiments a cytokine is a member of the extended IL-10 family (e.g.,IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, or IL-29). Where acytokine is referred to herein by number, all family members having thesame number are encompassed. For example, IL-17 refers to any or allIL-17 family members (e.g., IL-17A, IL-17B, IL-17C, IL-17D, IL17E,IL-17F).

In some embodiments a CPCA may be used to analyze one or more effects ofintracellular C3 activation. In some embodiments a CPCA may be used toanalyze the effects of inhibiting intracellular C3 activation. In someembodiments analysis is performed using isolated cells in culture, e.g.,using a composition comprising isolated cells and a CPCA (e.g., any ofthe compositions described above). A composition comprising isolatedcells and a CPCA is prepared. One or more biological activities orphenotypes of the cells is detected or measured. The nature or amount ofthe biological activity or phenotype is compared with a control. Asuitable control may be cells of the same type maintained under the sameconditions in the absence of the CPCA. In some embodiments a biologicalactivity comprises secretion of one or more biologically active agents,e.g., a protein or signaling molecule. In some embodiments a biologicalactivity comprises cytokine secretion, protease secretion, cellproliferation, or cell-mediated cytotoxicity.

In some embodiments a CPCA may be used to analyze one or more effects ofintracellular C3 activation using a non-human mammal that, eithernaturally or as a result of genetic engineering, expresses primate C3,e.g., human C3. In some embodiments the non-human animal is a mammal,e.g., a primate. In some embodiments the non-human animal is atransgenic rodent, e.g., a transgenic mouse or rat, wherein at leastsome cells of the transgenic rodent comprise a transgene that encodes aprimate C3, e.g., human C3. In some embodiments the rodent is alsotransgenic for any one or more additional primate complement components.In some embodiments the non-human animal is a mammal that has received agraft of primate cells, e.g., human cells, that express C3. In someembodiments the cells comprise hematopoietic cells, hepatic cells, orboth. In some embodiments the endogenous hematopoietic system and/orliver of the animal is ablated or removed. In some embodiments themammal has a humanized hematopoietic system or at least a humanizedimmune system. Methods for generating such mammals, e.g., mice, areknown. For example, the hu-HSC model is created by transplantation ofhuman hematopoietic stem cells (HSC), whereas the BLT mouse model isprepared by transplantation of human fetal liver, thymus and HSC. Insome embodiments the non-human animal is an animal model for a disease,e.g., a disease that affects humans. In some embodiments the disease isa complement-mediated disease.

In some embodiments a CPCA is administered one or more times to asubject. In some embodiments the subject has a complement-mediateddisease or is at increased risk of developing a complement-mediateddisease. In some embodiments the effect of the CPCA on development,severity, or progression of the disease or on the development, severity,or progression of one or more manifestations of the disease is assessed.The complement-mediated disease may be any of the complement-mediateddiseases described herein. For example, in some embodiments thecomplement-mediated disease is a respiratory disease, e.g., asthma orCOPD. In some embodiments the complement-mediated disease is aThl7-associated disease. In some embodiments a CPCA is administered totreat a complement-mediated disease.

In some embodiments human cells or non-human primate cells contactedwith a CPCA at a concentration and for a time that is sufficient toreduce intracellular C3 cleavage and/or reduce release of one or more C3cleavage product(s) by such cells by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% exhibit no more than a 20% cytoxicity(reduction in viability) as compared to the viability of suitablecontrol cells, e.g., no detectable cytotoxicity, less than a 1%reduction in viability, between 1% and 2.5%, 2.5% and 5%, 5% and 10%,10% and a 20% reduction in viability as compared to the viability ofsuitable control cells. In some embodiments mammalian cells (which maybe human cells, non-human primate cells, or non-primate mammalian cells)contacted with a CPCA at a concentration and for a time that issufficient to reduce intracellular C3 cleavage and/or reduce release ofone or more C3 cleavage product(s) by human cells of at least one celltype by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%exhibit no more than a 20% reduction in viability (rounded to thenearest tenth of a percent) as compared to the viability of suitablecontrol cells, e.g., no detectable cytotoxicity, less than a 1%reduction in viability, between 1% and 2.5%, 2.5% and 5%, 5% and 10%,10% and a 20% reduction in viability as compared to the viability ofsuitable control cells. Cells are typically maintained under normal cellculture conditions for the particular cells (e.g., 37 degrees C.,standard culture medium (except for the presence of the CPCA in the testcell medium) for the cells, etc. In some embodiments suitable controlcells are cells that are not contacted with the CPCA. Control cells aretypically closely matched with the cells that are contacted with theCPCA (“test cells”) and are maintained under similar conditions (butwithout the CPCA. For example, test cells and control cells may bederived from the same cell line, same subject (or genetically identicalsubject or subject from the same inbred non-human animal strain). Insome embodiments test and control cells are from the same cell culture.In some embodiments control cells may be contacted with the vehicle inwhich the CPCA is provided when contacted with test cells in order tocontrol for possible of the vehicle. In some embodiments control cellsmay be contacted with a compstatin analog of the same sequence as the CAmoiety in the CPCA. In some embodiments control cells are cultured andtheir viability tested as part of the same experiment in which viabilityof test cells is measured. In some embodiments a historical controlvalue, e.g., a value that was previously determined for the same celltype cultured under the same or essentially the same conditions in theabsence of the CPCA and the viability of which was measured using thesame viability assay may be used. In some embodiments a difference inviability between two or more groups, e.g., test cells and controlcells, test cells contacted with different CPCAs, test cells contactedwith the same CPCA at different concentrations and/or for different timeperiods, is considered to exist if such difference is statisticallysignificant with a p value of 0.05. In some embodiments a difference isconsidered to exist if is statistically significant with a p value of0.025. In some embodiments a difference is considered to exist if isstatistically significant with a p value of 0.01. In some embodimentsviability is considered to be reduced by a given percentage based on anindicator of viability, such as a result of a viability assay, where theassay measures a substance, property, or event that correlates with cellviability. In some embodiments a raw value may be converted into apercent viability or percent change in viability using a suitableconversion factor or standard curve.

In some embodiments a CPCA is contacted with cells at a concentration upto about 1 μm, up to about 5 μM, up to about 10 μM, up to about 20 μM,up to about 50 μM, or up to about 100 μM for a time period of up to 1hour (e.g., 15-60 minutes), 4 hours, 8 hours, 12 hours, 16 hours, 24hours or a time period up to 1, 2, 4, 8, 12, 16, 24, 48, 60, 72, 96,120, or 144 hours or a time period up to 7, 10, 14, 21, or 28 days.Cytotoxicity may be assessed using any of a variety of methods known inthe art. Examples of such methods include a cell counting assay, areplication labeling assay, a cell membrane integrity assay (which maymeasure release of intracellular contents such as various enzymes or maydetect entry across the cell membrane of a substance that would normallynot cross the membrane of a viable cell), a cellular ATP-based viabilityassay, a mitochondrial reductase activity assay, a caspase activityassay, an annexin V staining assay, a DNA content assay, a DNAdegradation assay, and a nuclear fragmentation assay. Exemplary assaysinclude BrdU, EdU, or H3-Thymidine incorporation assays; DNA contentassays using a nucleic acid dye such as Hoechst dye, DAPI, actinomycinD, 7-aminoactinomycin D or propidium iodide; cellular metabolism assayssuch as AlamarBlue, MTT, XTT, WST-8, and CellTitre Glo; dye exclusionassay (e.g., trypan blue exclusion assay), LDH release assay,cytoplasmic histone-associated DNA fragments (e.g., mono- and/oroligonucleosomes); PARP cleavage assay; TUNEL staining. Appropriatemethod(s) may be selected by one of ordinary skill in the art. Forexample, certain of the assays measure events associated with cellproliferation such as DNA synthesis and may reflect effects of an agenton both viability and proliferation. If non-proliferating cells are usedan assay that does not rely on measuring an indicator of cellproliferation may be selected. It will also be appreciated that certainassays are specifically appropriate for measuring apoptotic cell death.In some embodiments one or more assays collectively capable of detectingcell death regardless of mechanism by which cell death occurs (e.g.,whether due to necrosis or apoptosis) is used. In some embodiments acell metabolism assay is used.

In some embodiments cells are contacted with a CPCA in vitro at aconcentration and for a time period sufficient to reduce intracellularC3 cleavage and/or reduce release of one or more C3 cleavage product(s)by such cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 100% measured over a particular time interval or in response to astimulus, as compared with the amount of intracellular C3 cleavage oramount of release of such C3 cleavage product(s) that would occur oversuch time period or in response to such stimulus if the cells were notcontacted with the CPCA. In some embodiments the time interval may be,e.g., between 5 minutes and 48 hours, e.g., between 5 and 60 minutes,1-2 hours, 2-4 hours, 4-8 hours, 8-16 hours, 16 to 24 hours, 24 to 48hours. In some embodiments the time period may be at least 48 hours,e.g., 2-5 days, 5-10 days, etc. If culture medium is replaced during thetime period, additional CPCA may be added to the new medium. In variousembodiments the time interval over which intracellular C3 cleavageand/or release of one or more C3 cleavage product(s) is measured mayoverlap with, be within, be the same as, or follow the time periodduring which cells are exposed to the CPCA in the medium.

Activity of a CPCA may be assessed using any of a variety of methods invarious embodiments. In some embodiments cells that produce primate(e.g., human) C3 are contacted with a CPCA in vitro. In some embodimentsthe cells are washed, lysed, and the cell lysate is analyzed todetermine the amount (e.g., concentration) of intact C3, the amount oneor more C3 cleavage products (e.g., C3a, C3b, iC3b, C3d, etc.), and/orthe ratio of the amount of intact C3 to that of one or more C3 cleavageproducts. A reduction in the level of C3 cleavage product(s) as comparedwith the level of such product(s) in control cells not contacted withthe CPCA indicates that the CPCA was internalized, bound tointracellular C3, and inhibited its activation. Care may be taken duringthe washing step to remove CPCA that may have become physicallyassociated with the cell surface during the contacting step. In someembodiments the cells are washed and lysed, and intact C3 is isolated,e.g., by an affinity-based method such as immunoprecipitation using asuitable antibody (or other binding agent). The isolated C3 is assessedto determine the amount of bound CPCA and/or to determine the amount orfraction of C3 that does not have a CPCA molecule bound to it. The CPCAmay be detected using any of a variety of methods. For example, the CPCAmay be detected using an affinity-based detection method (e.g.,immunoblot, ELISA assay), by mass spectrometry, by liquidchromatography, or combinations thereof. C3 that does not have a CPCAmolecule bound to it may be detected using any appropriate method.

In some embodiments a functional assay is used. In some embodiments afunctional assay comprises contacting cells that produce primate (e.g.,human) C3 with a CPCA in vitro and detecting the ability of C3 from suchcells to subsequently participate in complement activation. In someembodiments, cell lysate or C3 isolated from the lysate is combined withcomplement components necessary for C3 activation and, in someembodiments, complement components needed for downstream events such asMAC formation. The complement components may be provided as C3-depletedserum. Complement activation capacity of the resulting composition maybe assessed using a suitable assay, e.g., a functional assay such as ahemolysis assay, as a measure of the amount of active C3. Complementactivation via any of the complement activation pathways may bemeasured. In some embodiments the amount of one or more products ofcomplement activation, e.g., C3 cleavage products, C5 cleavage products(e.g., C5a), or MAC that is generated is measured. Such measurement maybe performed using any suitable method, e.g., affinity-based, bioassay,etc. A reduction in complement activation capacity of C3 from cellscontacted with a CPCA as compared with the level of complementactivation capacity of C3 from control cells not contacted with the CPCAindicates that the CPCA was internalized, bound to intracellular C3, andinhibited its activation.

In some embodiments cells that produce primate C3 are contacted in vitrowith a CPCA. The cells are maintained in culture for a suitable timeperiod, e.g., between 15 minutes and 48 hours. In some embodiments thecells are exposed to a stimulus that would typically, in the absence ofa CPCA, cause them to release C3 cleavage products. The amount of one ormore C3 cleavage products (e.g., C3a or C3b) secreted by the cells intothe medium is measured. A reduction in the amount C3 cleavage productssecreted from cells that are or have been contacted with a CPCA ascompared with the amount of C3 cleavage products secreted by controlcells maintained under the same or substantially the same conditions butnot contacted with the CPCA indicates that the CPCA was internalized,bound to intracellular C3, and inhibited its activation.

A variety of methods of assessing complement components, complementactivation, and/or products of complement activation, and variousreagents (e.g., antibodies) that may be used in such methods, aredescribed in US Patent Application Publication 20120135430. Kits formeasuring total complement activity or for measuring one of more C3 orC3 cleavage products are commercially available, e.g., from Quidel, DRGInternational (e.g., Complement Activation Assay CAE (EIA-1530). In someembodiments C3a is measured using an ELISA assay or using surfaceplasmon resonance. In some embodiments C3a is measured using a bioassaythat measures one or more biological activities of C3a, such asactivation of a C3a receptor expressed by living cells. Activation of aC3aR may, for example, cause calcium influx into the cytoplasm,increased cytokine expression, degranulation, or chemotaxis of cellsthat express C3aR. Such effects may be detected in a variety of waysknown in the art such as use of calcium-sensitive dyes, reporter assays,etc.

VIII. Uses

Cell-penetrating compstatin analogs have a wide variety of uses. Withoutlimiting the invention in any way, certain uses of cell-penetratingcompstatin analogs, and related aspects of the invention, are describedherein. In some embodiments, a cell-penetrating compstatin analog isadministered to a subject suffering from or at risk ofcomplement-mediated damage to an organ, tissue, or cells. In someembodiments, a cell-penetrating compstatin analog is contacted with anorgan, tissue, or cells ex vivo and become internalized by at least somecells. The organ, tissue, or cells are introduced into a subject and areprotected at least in part from dysfunction or damage that may otherwisebe caused by intracellular complement activation and/or by secretion ofone or more complement activation products.

Certain uses of interest include: protecting various body structures,cells, tissues, organs from complement mediated dysfunction or damage inany of a variety of different complement-mediated disorders, reducingischemia/reperfusion (I/R) injury (e.g., in individuals suffering fromtrauma, vascular obstruction, myocardial infarction, or other situationsin which I/R injury may occur). The beneficial effects of inhibitingcomplement activation or formation or deposition of complementactivation products (e.g., C3a, C3b) inside or at the surface of cellsor other body structures are not limited to those resulting directlyfrom protection of the cells or structures themselves against directcomplement-mediated damage (e.g., preventing cell lysis). For example,inhibiting complement activation using a cell-penetrating compstatinanalog may reduce the generation of anaphylotoxins and resultinginflux/activation of cells such as neutrophils, mast cells, and/or otherpro-inflammatory events and/or reduce potentially damaging release ofintracellular contents, In thereby potentially having beneficial effectslocally, on remote cells, tissues, organ systems, or throughout thebody. In some embodiments a cell-penetrating compstatin analog may becontacted with a cell or administered to a subject having abnormallyhigh cathepsin expression or activity, e.g., abnormally high cathepsin Lexpression or activity.

Complement-Mediated Disorders

In some embodiments, a cell-penetrating compstatin analog is introducedinto the eye for treatment of an eye disorder such as age-relatedmacular degeneration (AMD), diabetic retinopathy, glaucoma, or uveitis.For example, a cell-penetrating compstatin analog may be introduced intothe vitreous cavity (e.g., by intravitreal injection), for treatment ofa subject at suffering from or at risk of AMD. In some embodiments acell-penetrating compstatin analog is introduced into the anteriorchamber, e.g., to treat anterior uveitis.

In some embodiments a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of an autoimmune disease,e.g., an autoimmune disease mediated at least in part by antibodies, Tcells, or other immune system cells or substances directed against oneor more self antigens.

In some embodiments a cell-penetrating compstatin analog may beintroduced into the synovial cavity, e.g., in a subject suffering fromarthritis (e.g., rheumatoid arthritis).

In some embodiments, a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of an intracerebralhemorrhage.

In some embodiments a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of myasthenia gravis.

In some embodiments a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of neuromyelitis optica (NMO),

In some embodiments a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of membranoproliferativeglomerulitis (MPGN), e.g., MPGN type I, MPGN type II, or MPGH type III.

In some embodiments a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of a neurodegenerativedisease. In some embodiments a cell-penetrating compstatin analog isused to treat a subject suffering from neuropathic pain or at risk ofdeveloping neuropathic pain. In some embodiments a cell-penetratingcompstatin analog is used to treat a subject suffering from or at riskof rhinosinusitis or nasal polyposis. In some embodiments acell-penetrating compstatin analog is used to treat a subject sufferingfrom or at risk of cancer. In some embodiments a cell-penetratingcompstatin analog is used to treat a subject suffering from or at riskof sepsis. In some embodiments a cell-penetrating compstatin analog isused to treat a subject suffering from or at risk of adult respiratorydistress syndrome.

In some embodiments a cell-penetrating compstatin analog is used totreat a subject suffering from or at risk of anaphylaxis or infusionreaction. For example, in some embodiments a subject may be pretreatedprior to, during, or after receiving a drug or a vehicle that may causeanaphylaxis or infusion reaction. In some embodiments a subject at riskof or suffering from anaphylaxis from a food (e.g., peanut, shellfish,or other food allergens), insect sting (e.g., bee, wasp), is treatedwith a cell-penetrating compstatin analog.

A cell-penetrating compstatin analog may be administered locally orsystemically, in various embodiments of the invention.

In some embodiments, a cell-penetrating compstatin analog is used totreat a respiratory disease, e.g., asthma or chronic obstructivepulmonary disease (COPD). The cell-penetrating compstatin analog may,for example, be administered to the respiratory tract by inhalation,e.g., as a dry powder or via nebulization, or may be administered byinjection, e.g., intravenously, intramuscularly, or subcutaneously, invarious embodiments. In some embodiments, a cell-penetrating compstatinanalog is used to treat severe asthma, e.g., asthma that is notsufficiently controlled by bronchodilators and/or inhaledcorticosteroids.

In some aspects, methods of treating a complement-mediated disorder,e.g., a chronic complement-mediated disorder, are provided, the methodscomprising administering a cell-penetrating compstatin to a subject inneed of treatment for the disorder. The long-acting compstatin analogmay be any long-acting compstatin analog described herein, in variousembodiments. In some aspects, methods of treating a Thl7-associateddisorder are provided, the methods comprising administering acell-penetrating complement inhibitor to a subject in need of treatmentfor the disorder.

In some aspects, a “chronic disorder” is a disorder that persists for atleast 3 months and/or is accepted in the art as being a chronicdisorder. In many embodiments, a chronic disorder persists for at least6 months, e.g., at least 1 year, or more, e.g., indefinitely. One ofordinary skill in the art will appreciate that at least somemanifestations of various chronic disorders may be intermittent and/ormay wax and wane in severity over time. A chronic disorder may beprogressive, e.g., having a tendency to become more severe or affectlarger areas over time. A number of chronic complement-mediateddisorders are discussed herein. A chronic complement-mediated disordermay be any chronic disorder in which complement activation (e.g.,excessive or inappropriate complement activation) is involved, e.g., asa contributing and/or at least partially causative factor. Forconvenience, disorders are sometimes grouped by reference to an organ orsystem that is often particularly affected in subjects suffering fromthe disorder. It will be appreciated that a number of disorders canaffect multiple organs or systems, and such classification(s) are in noway limiting. Furthermore, a number of manifestations (e.g., symptoms)may occur in subjects suffering from any of a number of differentdisorders. Non-limiting information regarding disorders of interestherein may be found, e.g., in standard textbooks of internal medicinesuch as Cecil Textbook of Medicine (e.g., 23rd edition), Harrison'sPrinciples of Internal Medicine (e.g., 17th edition), and/or standardtextbooks focusing on particular areas of medicine, particular bodysystems or organs, and/or particular disorders.

In some embodiments, a chronic complement-mediated disorder is aTh2-associated disorder. As used herein, a Th2-associated disorder is adisorder characterized by an excessive number and/or excessive orinappropriate activity of CD4+ helper T cells of the Th2 subtype (“Th2cells”) in the body or a portion thereof, e.g., in at least one tissue,organ, or structure. For example, there may be a predominance of Th2cells relative to CD4+ helper T cells of the Thl subtype (“Thl cells”)e.g., in at least one tissue, organ, or structure affected by adisorder. As known in the art, Th2 cells typically secretecharacteristic cytokines such as interleukin-4 (IL-4), interleukin-5(IL-5), and interleukin-13 (IL-13), while Thl cells typically secreteinterferon-γ (IFN-γ) and tumor necrosis factor β (TNF β). In someembodiments, a Th2-associated disorder is characterized by excessiveproduction and/or amount of IL-4, IL-5, and/or IL-13, e.g., relative toIFN-γ and/or TNF β e.g., in at least some at least one tissue, organ, orstructure

In some embodiments, a chronic complement-mediated disorder is a Thl7-associated disorder. In some aspects, as described in further detailin PCT/US2012/043845, filed Jun. 22, 2012, entitled “Methods of TreatingChronic Disorders with Complement Inhibitors”, complement activation andThl 7 cells participate in a cycle that involves dendritic cells andantibodies and that contributes to maintenance of a pathologicimmunologic microenvironment underlying a range of disorders. Withoutwishing to be bound by any theory, the pathologic immunologicmicroenvironment, once established, is self-sustaining and contributesto cell and tissue injury. In some aspects, cell-penetrating compstatinanalogs are of use to treat Thl7-associated disorders.

As used herein, a Thl7-associated disorder is a disorder characterizedby an excessive number and/or excessive or inappropriate activity ofCD4+ helper T cells of the Thl7 subtype (“Thl7 cells”) in the body or aportion thereof, e.g., in at least one tissue, organ, or structure. Forexample, there may be a predominance of Thl7 cells relative to Thland/or Th2 cells, e.g., in at least one tissue, organ, or structureaffected by a disorder. In some embodiments a predominance of Thl 7cells is a relative predominance, e.g., the ratio of Thl 7 cells to Thlcells and/or the ratio of Thl 7 cells to Th2 cells, is increasedrelative to normal values. In some embodiments the ratio of Thl 7 cellsto T regulatory cells (CD4⁺CD25⁺ regulatory T cells, also termed “Tregcells”), is increased relative to normal values. Formation of Thl 7cells and/or activation of Th 17 cells is promoted by various cytokines,e.g., interleukin 6 (IL-6), interleukin 21 (IL-21), interleukin 23(IL-23), and/or interleukin 1β (IL-Iβ). Formation of Thl 7 cellsencompasses differentiation of precursor T cells, e.g., naive CD4+ Tcells, towards a Thl 7 phenotype and their maturation into functionalThl 7 cells. In some embodiments, formation of Thl 7 cells encompassesany aspect of development, proliferation (expansion), survival, and/ormaturation of Thl 7 cells. In some embodiments, a Thl7-associateddisorder is characterized by excessive production and/or amount of IL-6,IL-21, IL-23, and/or IL-Iβ. Thl 7 cells typically secrete characteristiccytokines such as interleukin-17A (IL-17A), interleukin-17F (IL-17F),interleukin-21 (IL-21), and interleukin-22 (IL-22). In some embodiments,a Thl7-associated disorder is characterized by excessive productionand/or amount of a Thl 7 effector cytokine, e.g., IL-17A, IL-17F, IL-21,and/or IL-22. In some embodiments excessive production or amount of acytokine is detectable in the blood. In some embodiments excessiveproduction or amount of a cytokine is detectable locally, e.g., in atleast one tissue, organ or structure. In some embodiments aThl7-associated disorder is associated with a decreased number of Tregsand/or decreased amount of a Treg-associated cytokine. In someembodiments a Thl 7 disorder is any chronic inflammatory disease, whichterm encompasses a range of ailments characterized by self-perpetuatingimmune insults to a variety of tissues and that seem to be dissociatedfrom the initial insult that caused the ailment (which may be unknown).In some embodiments a Thl7-associated disorder is any autoimmunedisease. Many if not most “chronic inflammatory diseases” may in fact beauto-immune diseases. Examples of Thl 7-associated disorders includeinflammatory skin diseases such as psoriasis and atopic dermatitis;systemic scleroderma and sclerosis; inflammatory bowel disease (IBD)(such as Crohn's disease and ulcerative colitis); Behcet's Disease;dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis;meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis;rheumatoid arthritis (RA), Sjorgen's syndrome, multiple sclerosis,vasculitis; central nervous system (CNS) inflammatory disorders, chronichepatitis; chronic pancreatitis, glomerulonephritis; sarcoidosis;thyroiditis, pathologic immune responses to tissue/organ transplantation(e.g., transplant rejection); COPD, asthma, bronchiolitis,hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF),periodontitis, and gingivitis. In some embodiments a Thl7 disease is aclassically known auto-immune disease such as Type I diabetes orpsoriasis. In some embodiments a Thl7-associated disorder is age-relatedmacular degeneration.

In some embodiments, a chronic complement-mediated disorder is anIgE-associated disorder. As used herein, an “IgE-associated disorder” isa disorder characterized by excessive and/or inappropriate productionand/or amount of IgE, excessive or inappropriate activity of IgEproducing cells (e.g., IgE producing B cells or plasma cells), and/orexcessive and/or inappropriate activity of IgE responsive cells such aseosinophils or mast cells. In some embodiments, an IgE-associateddisorder is characterized by elevated levels of total IgE and/or in someembodiments, allergen-specific IgE, in the plasma of a subject and/orlocally.

In some embodiments, a chronic complement-mediated disorder ischaracterized by the presence of autoantibodies and/or immune complexesin the body, which may activate complement via, e.g., the classicalpathway. Autoantibodies may, for example, bind to self antigens, e.g.,on cells or tissues in the body. In some embodiments, autoantibodiesbind to antigens in blood vessels, skin, nerves, muscle, connectivetissue, heart, kidney, thyroid, etc. In some embodiments, a chroniccomplement-mediated disorder is not characterized by autoantibodiesand/or immune complexes.

In some embodiments, a chronic complement-mediated disorder is arespiratory disorder. In some embodiments, a chronic respiratorydisorder is asthma or chronic obstructive pulmonary disease (COPD). Insome embodiments, a chronic respiratory disorder is pulmonary fibrosis(e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury,allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis(also known as allergic alveolitis), eosinophilic pneumonia,interstitial pneumonia, sarcoid, Wegener's granulomatosis, orbronchiolitis obliterans. In some embodiments, the invention provides amethod of treating a subject in need of treatment for a chronicrespiratory disorder, e.g., asthma, COPD, pulmonary fibrosis,radiation-induced lung injury, allergic bronchopulmonary aspergillosis,hypersensitivity pneumonitis (also known as allergic alveolitis),eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener'sgranulomatosis, or bronchiolitis obliterans, the method comprisingadministering a cell-penetrating compstatin analog to a subject in needof treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is allergicrhinitis, rhinosinusitis, or nasal polyposis. In some embodiments, theinvention provides a method of treating a subject in need of treatmentfor allergic rhinitis, rhinosinusitis, or nasal polyposis, the methodcomprising administering a cell-penetrating compstatin analog to asubject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is adisorder that affects the musculoskeletal system. Examples of suchdisorders include inflammatory joint conditions (e.g., arthritis such asrheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis,spondyloarthropathies Reiter's syndrome, gout). In some embodiments, amusculoskeletal system disorder results in symptoms such as pain,stiffness and/or limitation of motion of the affected body part(s).Inflammatory myopathies include dermatomyositis, polymyositis, andvarious others are disorders of chronic muscle inflammation of unknownetiology that result in muscle weakness. In some embodiments a chroniccomplement-mediated disorder is myasthenia gravis. In some embodiments,the invention provides a method of treating any of the foregoingdisorders affecting the musculoskeletal system, the method comprisingadministering a cell-penetrating compstatin analog to a subject in needof treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is adisorder that affects the integumentary system. Examples of suchdisorders include, e.g., atopic dermatitis, psoriasis, pemphigus,systemic lupus erythematosus, dermatomyositis, scleroderma,sclerodermatomyositis, Sj5gren syndrome, and chronic urticaria. In someaspects, the invention provides a method of treating any of theforegoing disorders affecting the integumentary system, the methodcomprising administering a cell-penetrating compstatin analog to asubject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder affects thenervous system, e.g., the central nervous system (CNS) and/or peripheralnervous system (PNS). Examples of such disorders include, e.g., multiplesclerosis, other chronic demyelinating diseases, amyotrophic lateralsclerosis, chronic pain, stroke, allergic neuritis, Huntington'sdisease, Alzheimer's disease, and Parkinson's disease. In someembodiments, the invention provides a method of treating any of theforegoing disorders affecting the nervous system, the method comprisingadministering a cell-penetrating compstatin analog to a subject in needof treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder affects thecirculatory system. For example, in some embodiments the disorder is avasculitis or other disorder associated with vessel inflammation, e.g.,blood vessel and/or lymph vessel inflammation. In some embodiments, avasculitis is polyarteritis nodosa, Wegener's granulomatosis, giant cellarteritis, Churg-Strauss syndrome, microscopic polyangiitis,Henoch-Schonlein purpura, Takayasu's arteritis, Kawasaki disease, orBehcet's disease. In some embodiments, a subject, e.g., a subject inneed of treatment for vasculitis, is positive for antineutrophilcytoplasmic antibody (ANCA).

In some embodiments, a chronic complement-mediated disorder affects thegastrointestinal system. For example, the disorder may be inflammatorybowel disease, e.g., Crohn's disease or ulcerative colitis. In someembodiments, the invention provides a method of treating a chroniccomplement-mediated disorder that affects the gastrointestinal system,the method comprising administering a cell-penetrating compstatin analogto a subject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is athyroiditis (e.g., Hashimoto's thryoiditis, Graves' disease, post-partumthryoiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis,glomerulonephritis (e.g., membranoproliferative glomerulonephritis ormembranous glomerulonephritis), or panniculitis.

In some embodiments, the invention provides methods of treating asubject suffering from chronic pain, the methods comprisingadministering a cell-penetrating compstatin analog t to a subject inneed thereof. In some embodiments, a subject suffers from neuropathicpain. Neuropathic pain has been defined as pain initiated or caused by aprimary lesion or dysfunction in the nervous system, in particular, painarising as a direct consequence of a lesion or disease affecting thesomatosensory system. For example, neuropathic pain may arise fromlesions that involve the somatosensory pathways with damage to smallfibres in peripheral nerves and/or to the spino-thalamocortical systemin the CNS. In some embodiments, neuropathic pain arises from autoimmunedisease (e.g., multiple sclerosis), metabolic disease (e.g., diabetes),infection (e.g., viral disease such as shingles or HIV), vasculardisease (e.g., stroke), trauma (e.g., injury, surgery), or cancer. Forexample, neuropathic pain can be pain that persists after healing of aninjury or after cessation of a stimulus of peripheral nerve endings orpain that arises due to damage to nerves. Exemplary conditions of orassociated with neuropathic pain include painful diabetic neuropathy,post-herpetic neuralgia (e.g., pain persisting or recurring at the siteof acute herpes zoster 3 or more months after the acute episode),trigeminal neuralgia, cancer related neuropathic pain,chemotherapy-associated neuropathic pain, HIV-related neuropathic pain(e.g., from HIV neuropathy), central/post-stroke neuropathic pain,neuropathy associated with back pain, e.g., low back pain (e.g., fromradiculopathy such as spinal root compression, e.g., lumbar rootcompression, which compression may arise due to disc herniation), spinalstenosis, peripheral nerve injury pain, phantom limb pain,polyneuropathy, spinal cord injury related pain, myelopathy, andmultiple sclerosis. In certain embodiments of the invention acell-penetrating compstatin analog is administered to treat neuropathicpain in a subject with one or more of the afore-mentioned conditions.

In some embodiments, a chronic complement-mediated disorder is a chroniceye disorder. In some embodiments, the chronic eye disorder ischaracterized by macular degeneration, choroidal neovascularization(CNV), retinal neovascularization (RNV), ocular inflammation, or anycombination of the foregoing. Macular degeneration, CNV, RNV, and/orocular inflammation may be a defining and/or diagnostic feature of thedisorder. Exemplary disorders that are characterized by one or more ofthese features include, but are not limited to, macular degenerationrelated conditions, diabetic retinopathy, retinopathy of prematurity,proliferative vitreoretinopathy, uveitis, keratitis, conjunctivitis, andscleritis. Macular degeneration related conditions include, e.g.,age-related macular degeneration (AMD). In some embodiments, a subjectis in need of treatment for wet AMD. In some embodiments, a subject isin need of treatment for dry AMD. In some embodiments, a subject is inneed of treatment for geographic atrophy (GA). In some embodiments, asubject is in need of treatment for ocular inflammation. Ocularinflammation can affect a large number of eye structures such as theconjunctiva (conjunctivitis), cornea (keratitis), episclera, sclera(scleritis), uveal tract, retina, vasculature, and/or optic nerve.Evidence of ocular inflammation can include the presence ofinflammation-associated cells such as white blood cells (e.g.,neutrophils, macrophages) in the eye, the presence of endogenousinflammatory mediator(s), one or more symptoms such as eye pain,redness, light sensitivity, blurred vision and floaters, etc. Uveitis isa general term that refers to inflammation in the uvea of the eye, e.g.,in any of the structures of the uvea, including the iris, ciliary bodyor choroid. Specific types of uveitis include iritis, iridocyclitis,cyclitis, pars planitis and choroiditis. In some embodiments, a subjectis in need of treatment for geographic atrophy (GA). In someembodiments, the chronic eye disorder is an eye disorder characterizedby optic nerve damage (e.g., optic nerve degeneration), such asglaucoma.

As noted above, in some embodiments the chronic respiratory disease isasthma. Information regarding risk factors, epidemiology, pathogenesis,diagnosis, current management of asthma, etc., may be found, e.g., in“Expert Panel Report 3: Guidelines for the Diagnosis and Management ofAsthma”. National Heart Lung and Blood Institute. 2007.http://www.nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf. (“NHLBIGuidelines”; www.nhlbi.nih.gov/guidelines/asthma/asthgdln.htm), GlobalInitiative for Asthma, Global Strategy for Asthma Management andPrevention 2010 “GINA Report”) and/or standard textbooks of internalmedicine such as Cecil Textbook of Medicine (20th edition), Harrison'sPrinciples of Internal Medicine (17th edition), and/or standardtextbooks focusing on pulmonary medicine. Asthma is a chronicinflammatory disorder of the airways in which many cells and cellularelements play a role, such as, mast cells, eosinophils, T lymphocytes,macrophages, neutrophils, and epithelial cells Asthmatic individualsexperience recurrent episodes associated with symptoms such as wheezing,breathlessness (also termed dyspnea or shortness of breath), chesttightness, and coughing. These episodes are usually associated withwidespread but variable airflow obstruction that is often reversible,either spontaneously or with treatment. The inflammation also causes anassociated increase in the existing bronchial hyperresponsiveness to avariety of stimuli. Airway hyperresponsiveness (an exaggeratedbronchoconstrictor response to stimuli) is a typical feature of asthma.In general, airflow limitation results from bronchoconstriction andairway edema. Reversibility of airflow limitation may be incomplete insome patients with asthma. For example, airway remodeling can lead tofixed airway narrowing. Structural changes can include thickening of thesub-basement membrane, subepithelial fibrosis, airway smooth musclehypertrophy and hyperplasia, blood vessel proliferation and dilation,and mucous gland hyperplasia, and hypersecretion.

Individuals with asthma may experience exacerbations, which areidentified as events characterized by a change from the individual'sprevious status. Severe asthma exacerbations can be defined as eventsthat require urgent action on the part of the individual and his/herphysician to prevent a serious outcome, such as hospitalization or deathfrom asthma. For example, a severe asthma exacerbation may require useof systemic corticosteroids (e.g., oral corticosteroids) in a subjectwhose asthma is usually well controlled without OCS or may require anincrease in a stable maintenance dose. Moderate asthma exacerbations canbe defined as events that are troublesome to the subject, and thatprompt a need for a change in treatment, but that are not severe. Theseevents are clinically identified by being outside the subject's usualrange of day-to-day asthma variation.

Current medications for asthma are typically categorized into twogeneral classes: long-term control medications (“controllermedications”) such as inhaled corticosteroids (ICS), oralcorticosteroids (OCS), long-acting bronchodilators (LABAs), leukotrienemodifiers (e.g., leukotriene receptor antagonists or leukotrienesynthesis inhibitors, anti-IgE antibodies (omalizumab (Xolair®)),cromolyn and nedocromil, which are used to achieve and maintain controlof persistent asthma and quick-relief medications such as short-actingbronchodilators (SABAs), which are used to treat acute symptoms andexacerbations. For purposes of the present invention, these treatmentsmay be referred to as “conventional therapy”. Treatment of exacerbationsmay also include increasing the dose and/or intensity of controllermedication therapy. For example, a course of OCS can be used to regainasthma control. Current guidelines mandate daily administration ofcontroller medication or, in many cases, administration of multipledoses of controller medication each day for subjects with persistentasthma (with the exception of Xolair, which is administered every 2 or 4weeks).

A subject is generally considered to have persistent asthma if thesubject suffers from symptoms on average more than twice a week and/ortypically uses a quick relief medication (e.g., SABA) more than twice aweek for symptom control. “Asthma severity” can be classified based onthe intensity of treatment required to control the subject's asthma oncerelevant comorbidities have been treated and inhaler technique andadherence have been optimized (see, e.g., GINA Report; Taylor, D R, EurRespir J 2008; 32:545-554). The description of treatment intensity canbe based on the medications and doses recommended in the stepwisetreatment algorithm found in guidelines such as NHLBI Guidelines 2007,GINA Report, and their predecessors and/or in standard medicaltextbooks. For example, asthma can be classified as intermittent, mild,moderate, or severe as indicated in Table X, where “treatment” refers totreatment sufficient to achieve subject's best level of asthma control.(It will be understood that the categories of mild, moderate, and severeasthma in general imply persistent rather than intermittent asthma). Oneof ordinary skill in the art will appreciate that Table X is exemplary,and that not all of these medications will be available in allhealthcare systems, which may affect the assessment of asthma severityin some environments. It will also be appreciated that other emerging ornew approaches may affect the classification of mild/moderate asthma.However, the same principle, of mild asthma being defined by the abilityto achieve good control using very low-intensity treatment and severeasthma being defined by the requirement for high-intensity treatment,can still be applied. Asthma severity can also or alternately beclassified based on intrinsic intensity of the disease in the absence oftreatment (see, e.g., NHBLI Guidelines 2007). Assessment can be made onthe basis of current spirometry and the patient's recall of symptomsover the previous 2-4 weeks. Parameters of current impairment and futurerisk may be assessed and included in a determination of the level ofasthma severity. In some embodiments, asthma severity is defined asshown in FIG. 3.4(a), 3.4(b), 3.4(c) of the NHBLI Guidelines, forindividuals 0-4, 5-11, or >12 years of age, respectively.

TABLE X Treatment-based Asthma Classification Asthma ClassificationTreatment Intermittent SABA as needed (typically no more than twice aweek) Mild Low-dose ICS or other low-intensity treatment (e.g., LTRA,cromolyn, nedocromil, theophylline) Moderate Low to moderate dose ICSand LABA or other extra treatment Severe High-intensity treatment(high-dose ICS and LABA ± oral corticosteroids and/or other extratreatment)

“Asthma control” refers to the extent to which the manifestations ofasthma have been reduced or removed by treatment (whetherpharmacological or non-pharmacological). Asthma control can be assessedbased on factors such as symptom frequency, nighttime symptoms,objective measures of lung function such as spirometry parameters (e.g.,% FEVi of predicted, FEVi variability, requirement for use of SABA forsymptom control. Parameters of current impairment and future risk may beassessed and included in a determination of the level of asthma control.In some embodiments, asthma control is defined as shown in FIG. 4.3(a),4.3(b), or 4.3(c) of NHBLI Guidelines, for individuals 0-4, 5-11, or ≥12years of age, respectively.

In general, one of ordinary skill in the art can select an appropriatemeans of determining asthma severity level and/or degree of control, andany classification scheme considered reasonable by those of ordinaryskill in the art can be used.

In some embodiments of the invention, a subject suffering frompersistent asthma is treated with a cell-penetrating compstatin analog.In some embodiments, the subject suffers from mild or moderate asthma.In some embodiments, the subject suffers from severe asthma. In someembodiments, a subject has asthma that is not well controlled usingconventional therapy. In some embodiments, a subject has asthma that,when treated using conventional therapy, requires use of ICS in order tobe well controlled. In some embodiments, a subject has asthma that failsto be well controlled despite use of ICS. In some embodiments, a subjecthas asthma that, if treated using conventional therapy, would requireuse of OCS in order to be well controlled. In some embodiments, asubject has asthma that fails to be well controlled despite use of highintensity conventional therapy that includes OCS.

In some embodiments, the subject suffers from allergic asthma, which isthe case for most asthmatic individuals. In some embodiments, anasthmatic subject is considered to have allergic asthma if anon-allergic trigger for the asthma (e.g., cold, exercise) is not knownand/or is not identified in a standard diagnostic evaluation. In someembodiments, an asthmatic subject is considered to have allergic asthmaif the subject (i) reproducibly develops asthma symptoms (or worseningof asthma symptoms) following exposure to an allergen or allergen(s) towhich the subject is sensitive; (ii) exhibits IgE specific for anallergen or allergen(s) to which the subject is sensitive; (iii)exhibits a positive skin-prick test to an allergen or allergen(s) towhich the subject is sensitive; and/or (iv) exhibits other symptom(s) ofcharacteristic(s) consistent with atopy such as allergic rhinitis,eczema, or elevated total serum IgE. It will be appreciated that aspecific allergic trigger may not be identified but may be suspected orinferred if the subject experiences worsening symptoms in particularenvironments, for example.

Allergen challenge by inhalation is a technique that is widely used inevaluating allergic airway disease. Inhalation of allergen leads tocross-linking of allergen-specific IgE bound to IgE receptors on, e.g.,mast cells and basophils. Activation of secretory pathways ensues,resulting in release of mediators of bronchoconstriction and vascularpermeability. Individuals with allergic asthma may develop variousmanifestations following allergen challenge, e.g., early asthmaticresponse (EAR), late asthmatic response (LAR), airway hyperreactivity(AHR), and airway eosinophilia, each of which can be detected andquantified as known in the art. For example, airway eosiphophilia may bedetected as an increase in eosinophils in sputum and/or BAL fluid. TheEAR, sometimes referred to as the immediate asthmatic response (IAR), isa response to allergen challenge by inhalation that becomes detectableshortly after the inhalation, typically within 10 minutes (min) of theinhalation, e.g., as a decrease in FEVi. The EAR typically reaches amaximum within 30 min and resolves within 2-3 hours (h) post-challenge.For example, a subject may be considered to exhibit a “positive” EAR ifhis/her FEVi decreases by at least 15%, e.g., at least 20%, within thistime window relative to baseline FEVi (where “baseline” in this contextrefers to conditions before the challenge, e.g., conditions equivalentto the subject's usual condition when not experiencing an asthmaexacerbation and not exposed to allergic stimuli to which the subject issensitive). The late asthmatic response (LAR) typically starts between 3h and 8 h post-challenge and is characterized by cellular inflammationof the airway, increased bronchiovascular permeability, and mucussecretion. It is typically detected as a decrease in FEVi, which may begreater in magnitude than that associated with the EAR and potentiallymore clinically important. For example, a subject may be considered toexhibit a “positive” LAR if his/her FEVi decreases by at least 15%,e.g., at least 20%, relative to baseline FEVi within the relevant timeperiod as compared with baseline FEVi. A delayed airway response (DAR)may occur beginning between about 26 and 32 h, reaching a maximumbetween about 32 and 48 h and resolving within about 56 h after thechallenge (Pelikan, Z. Ann Allergy Asthma Immunol. 2010,104(5):394-404).

In some embodiments, the chronic respiratory disorder is chronicobstructive pulmonary disease (COPD). COPD encompasses a spectrum ofconditions characterized by airflow limitation that is not fullyreversible even with therapy and is usually progressive. Symptoms ofCOPD include dyspnea (breathlessness), decreased exercise tolerance,cough, sputum production, wheezing, and chest tightness. Persons withCOPD can experience episodes of acute (e.g., developing over course ofless than a week and often over the course of 24 hours or less)worsening of symptoms (termed COPD exacerbations) that can vary infrequency and duration and are associated with significant morbidity.They may be triggered by events such as respiratory infection, exposureto noxious particles, or may have an unknown etiology. Smoking is themost commonly encountered risk factor for COPD, and other inhalationalexposures can also contribute to development and progression of thedisease. The role of genetic factors in COPD is an area of activeresearch. A small percentage of COPD patients have a hereditarydeficiency of alpha-1 antitrypsin, a major circulating inhibitor ofserine proteases, and this deficiency can lead to a rapidly progressiveform of the disease.

Characteristic pathophysiologic features of COPD include narrowing ofand structural changes in the small airways and destruction of lungparenchyma (in particular around alveoli), most commonly due to chronicinflammation. The chronic airflow limitation observed in COPD typicallyinvolves a mixture of these factors, and their relative importance incontributing to airflow limitation and symptoms varies from person toperson. The term “emphysema” refers to enlargement of the air spaces(alveoli) distal to the terminal bronchioles, with destruction of theirwalls. It should be noted that the term “emphysema” is often usedclinically to refer to the medical condition associated with suchpathological changes. Some individuals with COPD have chronicbronchitis, which is defined in clinical terms as a cough with sputumproduction on most days for 3 months of a year, for 2 consecutive years.Further information regarding risk factors, epidemiology, pathogenesis,diagnosis, and current management of COPD may be found, e.g., in “GlobalStrategy for the Diagnosis, Management, and Prevention of ChronicObstructive Pulmonary Disease” (updated 2009) available on the GlobalInitiative on Chronic Obstructive Pulmonary Disease, Inc. (GOLD) website(www.goidcopd.org), also referred to herein as the “GOLD Report”, theAmerican Thoracic Society/European Respiratory Society Guidelines (2004)available on the ATS website at {circumflex over( )}vwvv.thoracic.org/clinical/copd-guidelmes/resources/copddoc.pdf.referred to herein as “ATC/ERS COPD Guidelines” and standard textbooksof internal medicine such as Cecil Textbook of Medicine (20^(th)edition), Harrison's Principles of Internal Medicine (17^(th) edition),and/or standard textbooks focusing on pulmonary medicine.

In some embodiments methods disclosed herein inhibit (interfere with,disrupt) the DC-Thl7-B-Ab-C-DC cycle described in PCT/US2012/043845. Forexample, administration of a complement inhibitor may break the cycle bywhich complement stimulates DC cells to promote the Thl7 phenotype. As aresult, the number and/or activity of Thl7 cells diminishes, which inturn reduces the amount of Thl7-mediated stimulation of B cells andpolyclonal antibody production. In some embodiments, these effectsresult in “resetting” the immunological microenvironment to a morenormal, less pathological state. In some embodiments, inhibiting theDC-Thl7-B-Ab-C-DC cycle has a disease-modifying effect. Without wishingto be bound by any theory, rather than merely treating symptoms of adisorder, inhibiting the DC-Thl7-B-Ab-C-DC cycle may interfere withfundamental pathologic mechanisms that may contribute to ongoing tissuedamage even when symptoms are well controlled and/or that may contributeto exacerbations of the disease. In some embodiments, inhibiting theDC-Thl7-B-Ab-C-DC cycle causes a chronic disorder to go into remission.In some embodiments, remission refers to a state of absence orsubstantial absence of disease activity in a subject with a chronicdisorder, with the possibility of return of disease. In some embodimentsremission may be sustained for a prolonged period of time (e.g., atleast 6 months, e.g., 6-12 months, 12-24 months, or more) in the absenceof continued therapy or with a reduced dose or increased dosinginterval. In some aspects, inhibition of complement may change theimmunological micro-environment of a tissue that is rich in Thl7 cellsand modify it into a micro-environment that is rich in regulatory Tcells (Tregs). Doing so could allow the immune system to “reset” itselfand go into a state of remission. In some embodiments, for example,remission may be sustained until occurrence of a triggering event. Atriggering event may be, for example, an infection (which may result inproduction of polyclonal antibodies that react both with an infectiousagent and a self protein), exposure to particular environmentalconditions (e.g., high levels of air pollutants such as ozone orparticulate matter or components of smoke such as cigarette smoke,allergens), etc. Genetic factors may play a role. For example,individuals having particular alleles of genes encoding complementcomponents may have a higher baseline level of complement activity, amore reactive complement system and/or a lower baseline level ofendogenous complement regulatory protein activity. In some embodimentsan individual has a genotype associated with increased risk of AMD. Forexample, the subject may have a polymorphism in a gene encoding acomplement protein or complement regulatory protein, e.g., CFH, C3,factor B, wherein the polymorphism is associated with an increased riskof AMD.

In some embodiments an immunologic microenvironment may becomeprogressively more polarized towards a pathological state over time,e.g., in a subject who has not yet developed symptoms of a chronicdisorder or in a subject who has developed the disorder and has beentreated as described herein. Such a transition may occur stochastically(e.g., due at least in part to apparently random fluctuations inantibody levels and/or affinity) and/or as a result of accumulated“sub-threshold” trigger events that are not of sufficient intensity totrigger a symptomatic outbreak of a disorder.

In some embodiments it is contemplated that a relatively short course ofa long-acting compstatin analog, e.g., between 1 week and 6 weeks, e.g.,about 2-4 week, may provide a long-lasting benefit. In some embodimentsa remission is achieved for a prolonged period of time, e.g., 1-3months, 3-6 months, 6-12 months, 12-24 months, or more. In someembodiments a subject may be monitored and/or treated prophylacticallybefore recurrence of symptoms. For example, a subject may be treatedprior to or upon exposure to a triggering event. In some embodiments asubject may be monitored, e g., for an increase in a biomarker, e.g., abiomarker comprising an indicator of Thl7 cells or Thl7 cell activity,or complement activation, and may be treated upon increase in the levelof such biomarker. See, e.g., PCT/US2012/043845 for further discussion.

Transplantation

Transplantation is a therapeutic approach of increasing importance,providing a means to replace organs and tissues that have been damagedthrough trauma, disease, or other conditions. Kidneys, liver, lungs,pancreas, and heart are among the organs that can be successfullytransplanted. Tissues that are frequently transplanted include bones,cartilage, tendons, cornea, skin, heart valves, and blood vessels.Pancreatic islet or islet cell transplantation is a promising approachfor treatment of diabetes, e.g., type I diabetes. For purposes of theinvention, an organ, tissue, or cell (or population of cells) that is betransplanted, is being transplanted, or has been transplanted may bereferred to as a “graft”. For purposes hereof, a blood transfusion isconsidered a “graft”.

Transplantation subjects the graft to a variety of damaging events andstimuli that can contribute to graft dysfunction and, potentially,failure. For example, ischemia-reperfusion (I/R) injury is a common andsignificant cause of morbidity and mortality in the case of many grafts(particularly solid organs) and can be a major determinant of likelihoodof graft survival. Transplant rejection is one of the major risksassociated with transplants between genetically different individualsand can lead to graft failure and a need to remove the graft from therecipient.

In some embodiments of the invention, a cell-penetrating compstatinanalog is used to protect a graft from complement-mediated damage. Acell-penetrating compstatin analog enters cells of the graft andinhibits complement activation. A graft can be contacted with acompstatin analog prior to, during, and/or after being transplanted, invarious embodiments of the invention. For example, prior totransplantation a graft removed from a donor can be contacted with aliquid comprising a compstatin analog. For example, the graft can bebathed in and/or perfused with the solution. In another embodiment, acompstatin analog is administered to a donor prior to removal of thegraft. In some embodiments, compstatin analog is administered to arecipient during and/or after the introduction of the graft. In someembodiments, a compstatin analog is delivered locally to thetransplanted graft. In some embodiments a compstatin analog isadministered systemically, e.g., intravenously.

The invention provides a composition comprising: (a) an isolated graft;and (b) a cell-penetrating compstatin analog. In some embodiments thecomposition further comprises a liquid solution suitable for contacting(e.g., suitable for rinsing, washing, bathing, perfusing, maintaining,or storing) a graft (e.g., an organ) such as an isolated graft that hasbeen removed from a donor and is awaiting transplantation to arecipient. In some embodiments the invention provides a compositioncomprising: (a) a liquid solution suitable for contacting a graft (e.g.,an organ); and (b) a cell-penetrating compstatin analog. The liquidsolution can be any liquid solution that is physiologically acceptableto the graft (e.g., appropriate osmotic composition, non-cytotoxic) andmedically acceptable in view of the subsequent introduction of the graftinto the recipient (e.g., preferably sterile or at least reasonably freefrom microorganisms or other contaminants) and compatible with thecompstatin analog (i.e., will not destroy the reactivity of thecompstatin analog). In some embodiments, a solution is any solution ownin the art for any such purposes. In some embodiments, a liquid solutionis Marshall's or Hyperosmolar Citrate (Soltran®, Baxter Healthcare),University of Wisconsin (UW) solution (ViaSpan™, Bristol Myers Squibb),Histidine Tryptophan Ketoglutarate (HTK) solution (Custodial®, KohlerMedical Limited), EuroCollins (Fresenius), and Celsior (SangstatMedical), Polysol, IGL-1, or AQIX® RS-1. Of course other solutions,e.g., containing equivalent or similar ingredients in the same ordifferent concentrations could be used within the scope ofphysiologically acceptable compositions. In some embodiments a solutiondoes not contain ingredient(s) with which the compstatin analog would beexpected to significantly react, and any solution may be modified ordesigned to lack such ingredients. In some embodiments, the compstatinanalog is present in the graft-compatible solution at a concentrationof, e.g., between 0.01 mg/ml and 100 mg/ml or may be added to thesolution to achieve such concentration.

In some embodiments, the invention provides a kit comprising: (a) acell-penetrating compstatin analog; and (b) a graft-compatible solutionor solid (e.g., powder) components thereof. The cell-penetratingcompstatin analog may be provided in solid form (e.g., powder) or atleast in part dissolved in a solution. In some embodiments thecompstatin analog and/or graft-compatible solution are provided inpredetermined amounts, so that when combined, a solution of appropriateconcentration for contacting a graft with the compstatin analog isproduced. In many embodiments the compstatin analog and graft-compatiblesolution or solid (e.g., powder) components thereof are in separatecontainers within the kit. In some embodiments the cell-penetratingcompstatin analog and components of a graft-compatible solution are bothprovided in solid (e.g., powder) form, either in separate containers ormixed. In some embodiments the kit comprises instructions for use, e.g.,instructions for adding a cell-penetrating compstatin analog to agraft-compatible solution and/or instructions for contacting a graftwith a cell-penetrating compstatin analog. Optionally the kit contains alabel approved by a government agency responsible for regulatingproducts used in transplantation, cell therapy, and/or bloodtransfusion.

The invention further provides a method of introducing acell-penetrating compstatin analog into cells of an isolated graftcomprising contacting the isolated graft with cell-penetratingcompstatin analog. The invention further provides an isolated grafthaving a cell-penetrating compstatin analog in at least some cells ofthe graft. Typically the isolated graft comprises multiple molecules ofcompstatin analog. In some embodiments, a graft is or comprises a solidorgan such as a kidney, liver, lung, pancreas, or heart. In someembodiments, a graft is or comprises bone, cartilage, fascia, tendon,ligament, cornea, sclera, pericardium, skin, heart valve, blood vessel,amniotic membrane, or dura mater. In some embodiments, a graft comprisesmultiple organs such as a heart-lung or pancreas-kidney graft. In someembodiments, a graft comprises less than a complete organ or tissue. Forexample, a graft may contain a portion of an organ or tissue, e.g., aliver lobe, section of blood vessel, skin flap, or heart valve. In someembodiments, a graft comprises a preparation comprising isolated cellsor tissue fragments that have been isolated from their tissue of originbut retain at least some tissue architecture, e.g., pancreatic islets.In some embodiments, a preparation comprises isolated cells that are notattached to each other via connective tissue, e.g., hematopoietic stemcells or progenitor cells derived from peripheral and/or cord blood, orwhole blood or any cell-containing blood product such as red blood cells(RBCs) or platelets. In some embodiments a graft is obtained from adeceased donor (e.g., a “donation after brain death” (DBD) donor or“donation after cardiac death” donor). In some embodiments, depending onthe particular type of graft, a graft is obtained from a living donor.For example, kidneys, liver sections, blood cells, are among the typesof grafts that can often be obtained from a living donor without unduerisk to the donor and consistent with sound medical practice.

In some embodiments, a graft is a xenograft (i.e., the donor andrecipient are of different species). In some embodiments a graft is anautograft (i.e., a graft from one part of the body to another part ofthe body in the same individual). In some embodiments, a graft is anisograft (i.e., the donor and recipient are genetically identical). Inmost embodiments, the graft is an allograft (i.e., the donor andrecipient are genetically non-identical members of the same species). Inthe case of an allograft, the donor and recipient may or may not begenetically related (e.g., family members). Typically, the donor andrecipient have compatible blood groups (at least ABO compatibility andoptionally Rh, Kell and/or other blood cell antigen compatibility). Therecipient's blood may have been screened for alloantibodies to the graftand/or the recipient and donor since the presence of such antibodies canlead to hyperacute rejection (i.e., rejection beginning almostimmediately, e.g., within several minutes after the graft comes intocontact with the recipient's blood). A complement-dependent cytoxicity(CDC) assay can be used to screen a subject's serum for anti-HLAantibodies. The serum is incubated with a panel of lymphcytes of knownHLA phenotype. If the serum contains antibodies against HLA molecules onthe target cells, cell death due to complement-mediated lysis occurs.Using a selected panel of target cells allows one to assign specificityto the detected antibody. Other techniques useful for determining thepresence or absence anti-HLA antibodies and, optionally, determiningtheir HLA specificity, include ELISA assays, flow cytometry assays,microbead array technology (e.g., Luminex technology). The methodologyfor performing these assays is well known, and a variety of kits forperforming them are commercially available.

In some embodiments a cell-penetrating compstatin analog inhibitscomplement-mediated rejection. For example, in some embodiments mayinhibit acute or chronic rejection. Without wishing to be bound by anytheory, inhibiting complement activation at the graft may reducecomplement fragment deposition on a cell surface and/or may inhibitleukocyte (e.g., neutrophil) infiltration, a contributor to graftfailure.

In some embodiments, a cell-penetrating compstatin analog may inhibitcomplement-mediated IR injury to a graft. As discussed further below, IRinjury can occur upon reperfusion of tissue whose blood supply has beentemporarily disrupted, as occurs in transplanted organs. Reducing/Rinjury would reduce the likelihood of acute graft dysfunction or reduceits severity, and reduce the likelihood of acute graft failure.

In some embodiments, a cell-penetrating compstatin analog inhibitschronic rejection and/or chronic graft failure. As used herein, “chronicrejection or graft failure” refers to rejection or failure occurring atleast 6 months post-transplant, e.g., between 6 months and 1, 2, 3, 4, 5years, or more post-transplant, often after months to years of goodgraft function. It is caused by a chronic inflammatory and immuneresponse against the graft. For purposes hereof, chronic rejection caninclude chronic allograft vasculopathy, a term used to refer to fibrosisof the internal blood vessels of the transplanted tissue.

In some embodiments, a cell-penetrating compstatin analog isadministered to a graft recipient to inhibit graft rejection and/orgraft failure.

Ischemia/Reperfusion Injury

Ischemia-reperfusion (I/R) injury is an important cause of tissue damagefollowing trauma and in other conditions associated with temporarydisruption of blood flow such as myocardial infarction, stroke, severeinfection, vascular disease, aneurysm repair, cardiopulmonary bypass,and transplantation.

In the setting of trauma, systemic hypoxemia, hypotension, and localinterruption of the blood supply resulting from contusions, compartmentsyndrome, and vascular injuries cause ischemia that damagesmetabolically active tissues. Restoration of the blood supply triggersan intense systemic inflammatory reaction that is often more harmfulthan the ischemia itself. Once the ischemic region is reperfused,factors that are produced and released locally enter the circulatorysystem and reach remote locations, sometimes causing significant damageto organs not affected by the original ischemic insult, such as thelungs and intestine, leading to single and multiple organ dysfunction.Complement activation occurs soon after reperfusion and is a keymediator of post-ischemic damage, both directly and through itschemoattractive and stimulatory effects on neutrophils. All three majorcomplement pathways are activated and, acting cooperatively orindependently, are involved in I/R related adverse events affectingnumerous organ systems. In some embodiments of the invention, acell-penetrating compstatin analog is administered to a subject who hasrecently (e.g., within the preceding 2, 4, 8, 12, 24, or 48 hours)experienced trauma, e.g., trauma that puts the subject at risk of I/Rinjury, e.g., due to systemic hypoxemia, hypotension, and/or localinterruption of the blood supply. In some embodiments thecell-penetrating compstatin analog may be administered intravascularly,optionally into a blood vessel that supplies an injured body part ordirectly to the body part. In some embodiments, the subject suffers fromspinal cord injury, traumatic brain injury, burn, and/or hemorrhagicshock.

In some embodiments, a cell-penetrating compstatin analog isadministered to a subject prior to, during, or after a surgicalprocedure, e.g., a surgical procedure that is expected to temporarilydisrupt blood flow to a tissue, organ, or portion of the body. Examplesof such procedures include cardiopulmonary bypass, angioplasty, heartvalve repair/replacement, aneurysm repair, or other vascular surgeries.The cell-penetrating compstatin analog may be administered prior to,after, and/or during an overlapping time period with the surgicalprocedure.

In some embodiments, a cell-penetrating compstatin analog isadministered to a subject who has suffered an MI, thromboembolic stroke,deep vein thrombosis, or pulmonary embolism. The cell-penetratingcompstatin analog may be administered in combination with a thrombolyticagent such as tissue plasminogen activator (tPA) (e.g., alteplase(Activase), reteplase (Retavase), tenecteplase (TNKase)), anistreplase(Eminase), streptokinase (Kabikinase, Streptase), or urokinase(Abbokinase). The cell-penetrating compstatin analog may be administeredprior to, after, and/or during an overlapping time period with thethrombolytic agent.

In some embodiments, a cell-penetrating compstatin analog isadministered to a subject to treat/R injury.

IX. Compositions and Administration

The invention provides a variety of compositions comprising acell-penetrating compstatin analog. In various embodiments, acomposition can have any feature or combination of features discussedherein so long as they are not mutually exclusive. The inventionprovides embodiments of such compositions, and methods of use thereof,in which the compstatin analog is any compstatin analog.

In some embodiments, a composition comprises a purified cell-penetratingcompstatin analog. Purification can be achieved using a variety ofapproaches that can be selected by one of ordinary skill in the artbased to achieve a desired degree of purity with respect to variouscomponents present in the composition prior to purification. Forexample, filtration, high performance liquid chromatography, affinitychromatography, and/or other approaches and combinations thereof can beused. In some embodiments, the composition comprises at least 80%, 85%,90%, 95%, 98%, 99%, or more cell-penetrating compstatin analog as apercentage of the total compstatin analog by weight. In someembodiments, the composition comprises at least 80%, 85%, 90%, 95%, 98%,99%, or cell-penetrating compstatin analog as a percentage of the totalcompstatin analog on a molar basis. In some embodiments, a compositionconsists or consists essentially of a cell-penetrating compstatinanalog. In some embodiments weight is dry weight.

In some aspects, the invention provides a pharmaceutical gradecomposition comprising a cell-penetrating compstatin analog. Thepharmaceutical grade composition can have any of the above-mentionedcharacteristics in terms of purity in various embodiments. Thepharmaceutical grade composition is sufficiently free of endotoxin,heavy metals, and unidentified and/or uncharacterized substances so asto be acceptable, without further purification, as a pharmaceuticalcomposition suitable for administration to a human subject or for themanufacture of a pharmaceutical composition to be administered to ahuman subject. In some embodiments, the pharmaceutical grade compositionis sterile.

Suitable preparations, e.g., substantially pure preparations of acell-penetrating compstatin analog or other active agent, may becombined with pharmaceutically acceptable carriers or vehicles, etc., toproduce an appropriate pharmaceutical composition. The term“pharmaceutically acceptable carrier or vehicle” refers to a non-toxiccarrier or vehicle that does not destroy the pharmacological activity ofthe compound with which it is formulated. One of skill in the art willunderstand that a carrier or vehicle is “non-toxic” if it is compatiblewith administration to a subject in an amount appropriate to deliver thecompound without causing undue toxicity. Pharmaceutically acceptablecarriers or vehicles that may be used in the compositions of thisinvention include, but are not limited to, water, physiological saline,Ringer's solution, sodium acetate or potassium acetate solution, 5%dextrose, and the like. The composition may include other components asappropriate for the formulation desired, e.g., as discussed herein.Supplementary active compounds, e.g., compounds independently useful fortreating a subject suffering from a complement-mediated disorder, canalso be incorporated into the compositions. The invention provides suchpharmaceutical compositions comprising a cell-penetrating compstatinanalog and, optionally, a second active agent useful for treating asubject suffering from a complement-mediated disorder.

In some embodiments, the invention provides a pharmaceuticallyacceptable composition suitable for administration to humans, packagedtogether with a label approved by a government agency responsible forregulating pharmaceutical agents, e.g., the U.S. Food & DrugAdministration. In some embodiments, the invention provides apharmaceutical kit or pack comprising: (a) a pharmaceutically acceptablecell-penetrating compstatin analog in solid form; (b) a pharmaceuticallyacceptable carrier or vehicle. Optionally the kit or pack containsinstructions for dissolving the cell-penetrating compstatin analog inthe carrier. In some embodiments a pharmaceutical kit or pack isprovided. The pack or kit comprises sufficient amount of pharmaceuticalcomposition for at least 1 dose, e.g., between 1 and 200 doses or anyintervening number or subrange. In some embodiments a pharmaceuticalpack or kit comprises one or more needles and, optionally, one or moresyringes. In some embodiments at least one prefilled syringe isprovided. In some embodiments one or more unit dosage forms orpremeasured aliquots are provided. In some embodiments instructions foradministration, which in some embodiments comprise instructions forself-administration, e.g., via subcutaneous injection, are provided.

A pharmaceutical composition can be administered to a subject by anysuitable route of administration including, but not limited to,intravenous, intramuscular, subcutaneously, by inhalation, by nasaldelivery, intrathecally, intracranially, intraarterially, orally,rectally, transdermally, intradermally, subdermally, etc. In someembodiments, a composition comprising a cell-penetrating compstatinanalog is administered intravenously. In some embodiments, a compositioncomprising a cell-penetrating compstatin analog is administeredintra-arterially. The composition can be administered locally, eitherinto the vascular system supplying an organ or tissue, orextra-vascularly in the vicinity of an organ or tissue. It will beunderstood that “administration” encompasses directly administering acompound or composition to a subject, instructing a third party toadminister a compound or composition to a subject, prescribing orsuggesting a compound or composition to a subject (e.g., forself-administration), self-administration, and, as appropriate, othermeans of making a compound or composition available to a subject.

Pharmaceutical compositions suitable for injectable use (e.g.,intravenous administration) or by pump or catheter typically includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Sterile solutions can be prepared byincorporating the compound in the required amount in an appropriatesolvent, optionally with one or a combination of ingredients such asbuffers such as acetates, citrates, lactates or phosphates; agents forthe adjustment of tonicity such as sodium chloride or dextrose;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid, glutathione, or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and othersuitable ingredients etc., as desired, followed by filter-basedsterilization. One of skill in the art will be aware of numerousphysiologically acceptable compounds that may be included in apharmaceutical composition. Other useful compounds include, for example,carbohydrates, such as glucose, sucrose, lactose; dextrans; amino acidssuch as glycine; polyols such as mannitol. These compounds may, forexample, serve as bulking agents and/or stabilizers, e.g., in a powderand/or when part of the manufacture or storage process involveslyophilization. Surfactant(s) such as Tween-80, Pluronic-F108/F68,deoxycholic acid, phosphatidylcholine, etc., may be included in acomposition, e.g., to increase solubility or to provide microemulsion todeliver hydrophobic drugs. pH can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide, if desired. The parenteralpreparation can be enclosed in ampoules, disposable syringes or infusionbags or multiple dose vials made of glass or plastic. Preferablysolutions for injection are sterile and acceptably free of endotoxin.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium andappropriate other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,methods of preparation can include vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient, e.g., from a previously sterile-filtered solution thereof.

Oral administration may be used in certain embodiments. Oralcompositions generally include an inert diluent or an edible carrier.For the purpose of oral therapeutic administration, the active compoundcan be incorporated with excipients and used in the

rm of tablets, troches, or capsules, e.g., gelatin capsules.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmacrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. A liquid compositioncan also be administered orally. Formulations for oral delivery mayincorporate agents to improve stability within the gastrointestinaltract and/or to enhance absorption.

For administration by inhalation, a compstatin analog may be deliveredin the form of an aerosol spray from a pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide. A metered dose inhaler or nebulizer may be used. The aerosolmay comprise liquid particles or dry aerosol (e.g., dry powders, largeporous particles, etc.).

For topical application, a compstatin analog may be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration include,but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, the pharmaceuticallyacceptable compositions can be formulated as a suitable lotion or creamcontaining a compstatin analog suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol,and water.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated may be used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished, e.g., through the use of nasal sprays or suppositories.For transdermal administration, the active compounds are typicallyformulated into ointments, salves, gels, or creams as generally known inthe art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In certain embodiments of the invention, a cell-penetrating compstatinanalog or other active compound is prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. For example, a cell-penetrating compstatin analog maybe incorporated into or encapsulated in a microparticle or nanoparticleformulation. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, poiyanhydrides, polyglycolic acid, collagen,polyorthoesters, polyethers, polylactic acid, PLGA, etc. Liposomes orother !ipid-hased particles can be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No. 4,522,811and/or other references listed herein. Depot formulations containing acell-penetrating compstatin analog may be-used. The cell-penetratingcompstatin analog is released from the depot over time, e.g., so as toprovide a therapeutic concentration for longer than if the compound wasadministered intravenously. In some aspects, a CRM confers depotproperties on a cell-penetrating compstatin analog. One of ordinaryskill in the art will appreciate that the materials and methods selectedfor preparation of a controlled release formulation, implant, etc.,should be such as to retain activity of the compound. In some aspects,described herein are particles, e.g., liposomes, nanoparticles,microparticles, comprising a cell-penetrating compstatin analog. Ananoparticle may have a diameter or longest dimension up to about 100tun, e.g., between about 10 nm and about 0.100 nm. A microparticle mayhave a diameter or longest dimension up to between about 100 nm about100 microns.

It will be appreciated that a compstatin analog, e.g., acell-penetrating compstatin analog, and/or additional active agent(s)can be provided as a pharmaceutically acceptable salt. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic and organic acids and bases. Examples of suitable acid saltsinclude acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate,glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxy ethanesulfonate, lactate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate,picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Also, pharmaceutically-acceptablesalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts, if appropriate depending on theidentity of the active agent.

It will be understood that the pharmaceutically acceptable carriers,compounds, and preparation methods mentioned herein are exemplary andnon-limiting. See, e.g., Remington: The Science and Practice ofPharmacy. 21st Edition. Philadelphia, Pa. Lippincott Williams & Wilkins,2005, for additional discussion of pharmaceutically acceptable compoundsand methods of preparing pharmaceutical compositions of various types.

A pharmaceutical composition can be administered in an amount effectiveto achieve a desired beneficial effect. In some embodiments, aneffective amount is sufficient to provide one or more of the followingbenefits: (i) reduction in at least one symptom or sign of acomplement-mediated disorder; (ii) increased quality of life; (iii)reduced hospitalization; (iv) reduced mortality. One of ordinary skillin the art will appreciate that the particular beneficial effect willdepend at least in part on various factors, such as the particulardisorder being treated. One of ordinary skill in the art will be awareof the symptoms and signs that may occur in subjects withcomplement-mediated disorders. Examples of symptoms and signs of variouscomplement disorders are provided herein. For example, in someembodiments, e.g., wherein a subject suffers from PNH or aHUS, abeneficial effect is a reduction in complement-mediated red blood celllysis. In some aspects, a beneficial effect is statistically significantand/or therapeutically meaningful within the judgement of one orordinary skill in the art.

In certain embodiments of the invention a pharmaceutical compositioncomprising a cell-penetrating compstatin analog is administeredparenterally. In some embodiments, the composition is administeredintravenously. In some embodiments, the composition is administered byintravenous injection. In some embodiments the composition isadministered as an IV bolus or an IV infusion. In some embodiments thecomposition is administered as an IV drip. In some embodiments thecomposition is administered as an IV bolus followed by an IV infusion orIV drip. In some embodiments an IV infusion is administered over about1, 2, 3, 4, 5, 15, 20, 30, 60, or 120 minutes. In some embodiments an IVdrip is administered over more than about 60 minutes, e.g., over about1, 2, 3, or more hours. In some embodiments, a total amount of betweenabout 0.1 mg/kg/day and about 2,000 mg/kg/day of compstatin analog isadministered, e.g., between about 1 mg/kg/day and about 1,000 mg/kg/day,e.g., between about 5 mg/kg/day and about 500 mg/kg/day. In someembodiments, a total amount of between about 10 mg/kg/day and about 100mg/kg/day of compstatin analog is administered, e.g., between about 10mg/kg/day and about 50 mg/kg/day e.g., between about 10 mg/kg/day andabout 20 mg/kg/day. It will be appreciated that a variety of differentdosing regimens could be used to administer a desired total dailyamount. For example, a desired amount of compstatin analog could beadministered in a single administration or in multiple administrations,e.g., during a 24 hour period. For example, a subject could receive twoor more doses within a 24 hour period, which doses could be administeredover the same length of time or over different lengths of time. In someembodiments, a cell-penetrating compstatin analog is administered attime intervals greater than 24 hours. For example, doses could beadministered on average every other day, every 3-4 days, weekly, everyother week, etc., in various embodiments. In some embodiments,covalently attached, long-acting, or targeted compstatin analogs protectcells, tissues, organs, for a period of weeks or months without need forretreatment. For example, subjects may be maintained with retreatment atintervals of between 1-2 weeks, 2-4 weeks, 4-6 weeks, 6-8 weeks, or evenlonger. In some embodiments subcutaneous administration is used toadminister at least some doses. For example, administration ofapproximately 0.1-5 mg/kg/day, e.g., about 0.5-2 mg/kg/day iscontemplated in some embodiments, e.g., in a volume of about 0.25 ml-2mL, e.g., a volume of about 1 ml. In some embodiments the concentrationis about 50 mg/ml to about 300 mg/ml, e.g., about 50 mg/ml-about 100mg/ml or about 100 mg/ml-about 200 mg/ml. In some embodimentsadministration is daily. In some embodiments administration is 1 or 2times per day. In some embodiments a cell-penetrating compstatin analogis administered using a therapeutically effective amount to a subject,wherein such administration results in blood concentrations of thecompound that achieve a level above at least 4 μM, at least 5 μM, atleast 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM,at least 11 μM, at least 12 μM, or at least 13 μM, at least 14 μM, atleast 15 μM, at least 16 μM, at least 18 μM, or at least about 20 μM, orat least about 25 μM or within any range between 4 μM and about 15 μM orabout 20 μM or about 25 μM. In some embodiments such level is maintainedfor at least about 24 hours, or at least about 48 hours, or at leastabout 72 hours, or at least about 96 hours, or at least about 120 hours,or at least about 144 hours, which may be achieved one or more IVinjections, infusion, subcutaneous injections, for example. Sustainedlevels may be achieved for longer, e.g., up to about 10 days, 12 days,14 days, or more.

It will be understood that there may be an initial treatment phaseduring which treatment is more frequent and/or in which higher doses areadministered. After that, lower doses and/or less frequent dosing may beused. In some embodiments treatment is started using rv administrationand then switched to subcutaneous, intramuscular, or intradermal formaintenance therapy. Depending on the disease, treatment may continue atintervals for, e.g., months, years, or indefinitely. Appropriate dosesand dosing regimen depend at least in part upon the potency andhalf-life of the compstatin analog (or other active agent), and mayoptionally be tailored to the particular recipient, for example, throughadministration of increasing doses until a preselected desired responseis achieved, such as a desired degree of complement inhibition and/orcell protection. If desired, the specific dose level for any particularsubject may be selected based at least in part upon a variety of factorsincluding the activity of the specific compound employed, the particularcondition being treated, the age, body weight, general health, route ofadministration, the rate of excretion, any drug combination, and/or thedegree of complement protein expression or activity measured in one ormore samples obtained from the subject.

The invention encompasses administration of a cell-penetratingcompstatin analog in combination with additional therapy. Suchadditional therapy may include administration of any agent(s) used inthe art or potentially useful for treating a subject suffering from thedisease. In any embodiment or aspect a cell-penetrating compstatinanalog may be administered in combination with a compstatin analoglacking a cell penetrating moiety, a long-acting, targeted, orcell-reactive compstatin analog, or a combination thereof. Long-acting,targeted, or cell-reactive compstatin analogs are described in PCT/USUS12/37648; U.S. Ser. No. 61/727,094; and/or U.S. Ser. No. 61/727,093.

When two or more therapies (e.g., compounds or compositions) are used oradministered “in combination” with each other, they may be given at thesame time, within overlapping time periods, or sequentially (e.g.,separated by up to 2-4 weeks in time), in various embodiments of theinvention. They may be administered via the same route or differentroutes. In some embodiments, the compounds or compositions areadministered within 48 hours of each other. In some embodiments, acell-penetrating compstatin analog analog can be given prior to or afteradministration of the additional compound(s), e.g., sufficiently closein time that the compstatin analog and additional compound(s) arepresent at useful levels within the body at least once. In someembodiments, the compounds or compositions are administered sufficientlyclose together in time such that no more than 90% of the earlieradministered composition has been metabolized to inactive metabolites oreliminated, e.g., excreted, from the body, at the time the secondcompound or composition is administered.

In some embodiments, a composition that includes both a cell-penetratingcompstatin analog and additional compound(s) is administered.

Example 1: Development of Cell Penetrating Compstatin Analogs

Three compounds, each of which incorporated the amino acid sequencelisted as SEQ ID NO: 28 and a different cell penetrating peptide, weresynthesized and designated as CPCA28-1, CPCA28-2, and CPCA28-3. Briefly,amino acids (including AEEAc) were obtained as Fmoc-protected aminoacids, in which the a-amino group of each amino acid was protected withFmoc. Side chain functional groups were also blocked with variousappropriate protective groups. Synthesis was accomplished following thesolid phase methodology described by Merrifield (J. Amer. Chem. Soc. 85,2149 (1963)). Chain assembly was performed on solid phase, at theconclusion of which the N-terminus was acetylated (in the case ofCPCA28-2 and CPCA28-3); the peptide was then cleaved from the solidphase and simultaneously deprotected via acidolysis using TFA andamidated. The linear peptide was then oxidized and purified. Compoundswere provided as acetate salts. Sequences are presented below:

CPCA28-1: (SEQ ID NO: 68)H-D-Arg-D-Arg-D-Arg-D-Gln-D-Arg-D-Arg-D-Lys-D-Lys-D-Arg-AEEAc-Ile-Cys-Val-Trp(Me)-Gin-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-M¾ (Trp(Me) ═ 1-methyl-1-tryptophan; “D” represents D amino acids) CPCA28-2: (SEQ ID NO: 69){circumflex over ( )}c-YARAAARQARAG-AEEAc-IC*V-(1-methyl-1-tryptophan)QDWGAHPvC*T-NH₂ CPCA28-3:  (SEQ ID NO: 70) {circumflex over( )}c-AAVALLPAVLLALLAP-AEEAc-IC* V(1-methyl-1-tryptophan)QDWGAHPvC*T-NH₂

To confirm that the compounds retains ability to inhibit C3 activation,inhibitory activity of the synthesized compounds is assessed bymeasuring the effect of the compounds on complement activation via theclassical pathway using a standard complement inhibition assay. Theprotocol measures C3b deposition in an ELISA format. C3b depositionmonitored using this method is generated through complement activated bythe classical pathway. Briefly, 96-well plates are coated with BSA.Human plasma, chicken ovalbumin (OVA), polyclonal anti-OVA antibodiesand compound being tested (referred to as “drug”) are added andincubated, followed by addition of Anti-human C3 HRP-conjugatedantibody. After an additional incubation, substrate is added and signaldetected. Details of the protocol are as follows:

Protocol for Classical Complement Inhibition Assay

Materials:

-   -   Ninety-six well plate (polystyrene plate, Thermo Scientific,        9205)    -   Chicken OVA (Sigma A5503-5G)    -   Rabbit anti-chicken OVA (Abeam abl221)    -   Blocking buffer (Startingblock buffer, Thermo Scientific 37538)    -   Veronal Buffer (5× concentration, Lonza 12-624E)    -   Human plasma (collected with Lepirudin at 50 ug/ml final        concentration)    -   Goat anti-human C3 HRP-conjugated Ab (MP Biomedicals, 55237)    -   Tween-20 Wash Buffer (0.05% Tween 20-PBS buffer)    -   TMB (Peroxidase substrate, BD 555214)—1:1 mixture of BD        51-2607KC and 51-2606KC.    -   1M H₂S0₄

Protocol:

-   -   1. Add 100 ul/well of 1% chicken OVA (in PBS)    -   2. Incubate overnight @ 4° C. or room temperature for 1-2 hr.    -   3. Remove by shaking and tapping the plate.    -   4. Block by adding 200 ul of blocking buffer    -   5. Incubate for 1 h at room temp    -   6. Remove by shaking and tapping the plate    -   7. Add 100 ul of 1:1000 dilution of Polyclonal anti-chicken OVA        in blocking buffer    -   8. Incubate for 1h at room temp    -   9. Wash twice with wash buffer    -   10. Add 50 ul VB⁺⁺ to wells #2 to 12    -   11. Add 1OO ul of starting drug dilution (2× in VB⁺⁺) to well 1.    -   12. Serially dilute (1:2) the drug from wells 1 to 10 as follow        -   a. Take 50 ul of solution from the originating well        -   b. Add this to the next well        -   c. Mix by pipetting several times        -   d. Repeat up to well #10    -   Note: from well #10 remove 50 ul and discard.    -   13. Add 50 ul of 2× plasma (1:37.5 dilution of original plasma)        dilution to wells 1 to 11    -   14. Incubate for 1h    -   15. Wash with wash buffer    -   16. Add lOO ul of 1/1000 dilution of anti-C3-HRP Ab in blocking        buffer    -   17. Incubate for 1h    -   18. Wash with wash buffer    -   19. Add lOO ul of TMB to all wells    -   20. Incubate for 5-10 min in dark    -   21. Add 50 ul 1M H₂S0₄    -   22. Read the plate at 450 nm

VB⁺⁺ Formula:

Barbital   5 mM NaCl 72.5 mM MgCl₂  0.5 mM CaC¾ 0.15 mM PH 7.4

Veronal Buffer (5X) Prod # MW For 500 ml 9 mM Sodium Barbitone SigmaB0500 206.17 927 mg 15.5 mM diethylbarbituric acid Sigma B0375 184.191.42 grams

Mg-Cl2 (200X) Prod # MW For 50 ml 100 mM MgCl₂—6H₂O Sigma M0250 203.301.00 gram

CaCl2 (500x) Prod # MW For 50 ml 75 mM CaCl₂ Sigma C7902 147.01 551.28mgTo prepare 50 ml of working buffer:

-   -   Weight 210 mg NaCl    -   Add 10 ml of 5× VB    -   Add lOO ul of CaCl₂ (500×)    -   Add 250 ul MgCl (200×)    -   Adjust volume to 50 ml with H₂0    -   Adjust pH to 7.4

Data is analyzed using GraphPad Prism5 software. Data sets from eachexperiment were normalized to percent activation compared to the 100%activation control corresponding to the well to which no compound isadded. Drug concentration values (X values) are transformed to theirlogarithms, and percent activation (Pa) (Y values) was transformed topercent inhibition (Pi) using the following formula Pi=100−Pa(Yi=100−Ya). The percent inhibition is plotted against the drugconcentration and the resulting data set was fit to a sigmoidal-doseresponse function [Y=Bottom+(Top−Bottom)/(1+10 ((Log EC−X)))]. IC5₀values were obtained from the fit parameters.

Example 2: Activity of Cell-Penetrating Compstatin Analogs in In VitroAssay

CPCA28-1, CPCA28-2, and CPCA28-3 are individually contacted in vitrowith C3 in the presence of cathepsin L. The ability of each compound toinhibit cathepsin L-mediated cleavage of C3 is assessed, e.g., by ELISAfor C3a. Various concentrations of compound are tested.

Example 3: Activity of Cell-Penetrating Compstatin Analogs in Cell-BasedAssay

CPCA28-1, CPCA28-2, and CPCA28-3 are individually contacted in vitrowith CD4+ Tcells. The T cells are activated (e.g., by contacting themwith antibody to CD3 or with antibody to CD3 and antibody to CD28).Ability of each compound to inhibit release of C3a and/or to inhibitdeposition of C3b on cell surfaces is assessed, e.g., using appropriateantibodies, e.g., with ELISA or flow cytometry. Various concentrationsof compound are tested.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. It will be appreciatedthat the invention is in no way dependent upon particular resultsachieved in any specific example or with any specific embodiment.Articles such as “a”, “an” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. For example, and withoutlimitation, it is understood that where claims or description indicatethat a residue at a particular position may be selected from aparticular group of amino acids or amino acid analogs, the inventionincludes individual embodiments in which the residue at that position isany of the listed amino acids or amino acid analogs. The invention alsoincludes embodiments in which more than one, or all of the group membersare present in, employed in, or otherwise relevant to a given product orprocess. Furthermore, it is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims or from the description above isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more elements,limitations, clauses, or descriptive terms, found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods ofadministering the composition according to any of the methods disclosedherein, and methods of using the composition for any of the purposesdisclosed herein are included within the scope of the invention, andmethods of making the composition according to any of the methods ofmaking disclosed herein are included within the scope of the invention,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Methods of treating a subject can include a step of providing asubject in need of such treatment (e.g., a subject who has had, or is atincreased risk of having, a disease), a step of diagnosing a subject ashaving a disease and/or a step of selecting a subject for treatment witha cell-penetrating compstatin analog. Where elements are presented aslists, it is to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. Forpurposes of conciseness only some of these embodiments have beenspecifically recited herein, but the invention includes all suchembodiments. It should also be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. Discussion of various diseases,disorders, and conditions under various headings herein is forconvenience and is not intended to limit the invention.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise. Any particular embodiment, aspect,element, feature, etc., of the present invention may be explicitlyexcluded from the claims even if such exclusion is not set forthexplicitly herein. For example, any compstatin analog, functional group,linking portion, clearance-reducing moiety, disease, or indication canbe explicitly excluded.

1. A compound comprising a compstatin analog moiety and a cellpenetrating moiety (CPM).
 2. The compound of claim 1, wherein the CPMcomprises a cell penetrating peptide.
 3. The compound of claim 1,wherein the CPM comprises a cationic cell penetrating peptide.
 4. Thecompound of claim 1, wherein the compstatin analog moiety is linked tothe CPM via a linker. 5-6. (canceled)
 7. The compound of claim 1,wherein the CPM comprises or consists of a cell penetrating peptide thesequence of which comprises or consists of or is derived from a sequenceset forth in Table
 2. 8. The compound of claim 1, wherein wherein thecompstatin analog moiety comprises a cyclic peptide having a coresequence of X′aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X′aa and Xaa areselected from Trp and analogs of Trp.
 9. The compound of claim 1,wherein the compstatin analog moiety comprises a cyclic peptide having acore sequence of X′aa-Gln -Asp-Xaa-Gly-X″aa (SEQ ID NO: 4), where X′aaand Xaa are each independently selected from Trp and analogs of Trp, andX″aa is selected from His, Ala, single methyl unbranched amino acids,Phe, Trp, and analogs of Trp.
 10. (canceled)
 11. The compound of claim1, wherein the compstatin analog is a compound that comprises a cyclicpeptide having a sequence ofX′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X″aa1-X″aa2-X″aa3-X″aa4-X″aa5(SEQ ID NO: 5), where X′aa4 and Xaa are selected from Trp and analogs ofTrp, wherein X′aa1, X′aa2, X′aa3, X″aa1, X″aa2, X″aa3, X″aa4, and X″aa5,are independently selected from among amino acids and amino acidanalogs, wherein the peptide is cyclized via a bond between X′aa2 andX″aa4. 12-13. (canceled)
 14. The compound of claim 1, wherein thecompstatin analog is a compound that comprises a cyclic peptide having asequence: (SEQ ID NO: 6) Xaa1 - Cys -Val - Xaa2 - Gln - Asp - Xaa2* -Gly - Xaa3 - His - Arg - Cys - Xaa4;

wherein: Xaa1 is Ile, Val, Leu, B¹—Ile, B¹—Val, B¹-Leu or a dipeptidecomprising Gly-Ile or B¹-Gly-Ile, and B¹ represents a first blockingmoiety; Xaa2 and Xaa2* are independently selected from Trp and analogsof Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog ofTrp; Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected fromThr-Ala and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein acarboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Ala, orAsn optionally is replaced by a second blocking moiety B²; and the twoCys residues are joined by a disulfide bond.
 15. The compound of claim14, wherein Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptidecomprising Gly-Ile or Ac-Gly-Ile; Xaa2 and Xaa2* are independentlyselected from Trp and analogs of Trp; Xaa3 is His, Ala or an analog ofAla, Phe, Trp, or an analog of Trp; Xaa4 is L-Thr, D-Thr, Ile, Val, Gly,a dipeptide selected from Thr-Ala and Thr-Asn, or a tripeptidecomprising Thr-Ala-Asn, wherein a carboxy terminal —OH of any of theL-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn optionally is replaced by —NH2.16. (canceled)
 17. The compound of claim 14, wherein Xaa2 is an analogof Trp comprising a substituted or unsubstituted bicyclic aromatic ringcomponent or two or more substituted or unsubstituted monocyclicaromatic ring components.
 18. (canceled)
 19. The compound of claim 14,wherein Xaa2 is an analog of Trp comprising a lower alkoxy or loweralkyl substituent at the 1 or 5 position of tryptophan or a halogensubstituent at the 5 or 6 position of tryptophan.
 20. The compound ofclaim 19, wherein Xaa2 is an analog of Trp comprising a lower alkoxy orlower alkyl substituent at the 1 or 5 position of tryptophan or ahalogen substituent at the 5 or 6 position of tryptophan and Xaa2* isTrp.
 21. The compound of claim 1, wherein the compstatin analogcomprises a cyclic peptide having a sequence selected from the groupconsisting of: SEQ ID NOs: 9-36.
 22. The compound of claim 1, whereinthe compstatin analog comprises a cyclic peptide having a sequenceselected from the group consisting of: SEQ ID NOs: 14, 21, 28, 29, 32,33, 34, or
 36. 23-32. (canceled)
 33. A method of inhibiting activationof primate complement component 3 (C3) comprising contacting said C3with a compound of claim
 1. 34-35. (canceled)
 36. A method of reducingthe sensitivity of a primate cell or subject to a complement activationstimulus, the method comprising contacting the primate cell or subjectwith the compstatin analog of claim
 1. 37. The method of claim 36,wherein the cell or subject is human.
 38. The method of claim 37,wherein the method comprises administering the compstatin analog to asubject.
 39. A method of treating a subject in need of treatment for acomplement-mediated disorder, the method comprising administering thecompstatin analog of claim 1 to the subject. 40-43. (canceled)
 44. Acomposition comprising (a) an isolated cell, tissue, or organ; and (b) acell penetrating compstatin analog.
 45. The composition of claim 44,wherein the isolated cell is a liver cell, endothelial cell, epithelialcell, or immune system cell.
 46. A method of characterizing acell-penetrating compstatin analog (CPCA), the method comprising: (a)contacting a cell that expresses primate C3 with a CPCA in vitro; and(b) assessing the activity of the CPCA.
 47. The method of claim 46,wherein assessing the activity of the CPCA comprises exposing the cellto a stimulus that would, in the absence of a CPCA, cause release of C3cleavage products from the cell; and measuring the amount of one or moreC3 cleavage products released from the cell.